Abstract
Preterm birth, the leading cause of perinatal morbidity and mortality, is associated with increased risk of short- and long-term adverse outcomes. For women identified as at risk for preterm birth attributable to a sonographic short cervix, the determination of imminent delivery is crucial for patient management. The current study aimed to identify amniotic fluid (AF) proteins that could predict imminent delivery in asymptomatic patients with a short cervix. This retrospective cohort study included women enrolled between May 2002 and September 2015 who were diagnosed with a sonographic short cervix (< 25 mm) at 16–32 weeks of gestation. Amniocenteses were performed to exclude intra-amniotic infection; none of the women included had clinical signs of infection or labor at the time of amniocentesis. An aptamer-based multiplex platform was used to profile 1310 AF proteins, and the differential protein abundance between women who delivered within two weeks from amniocentesis, and those who did not, was determined. The analysis included adjustment for quantitative cervical length and control of the false-positive rate at 10%. The area under the receiver operating characteristic curve was calculated to determine whether protein abundance in combination with cervical length improved the prediction of imminent preterm delivery as compared to cervical length alone. Of the 1,310 proteins profiled in AF, 17 were differentially abundant in women destined to deliver within two weeks of amniocentesis independently of the cervical length (adjusted p-value < 0.10). The decreased abundance of SNAP25 and the increased abundance of GPI, PTPN11, OLR1, ENO1, GAPDH, CHI3L1, RETN, CSF3, LCN2, CXCL1, CXCL8, PGLYRP1, LDHB, IL6, MMP8, and PRTN3 were associated with an increased risk of imminent delivery (odds ratio > 1.5 for each). The sensitivity at a 10% false-positive rate for the prediction of imminent delivery by a quantitative cervical length alone was 38%, yet it increased to 79% when combined with the abundance of four AF proteins (CXCL8, SNAP25, PTPN11, and MMP8). Neutrophil-mediated immunity, neutrophil activation, granulocyte activation, myeloid leukocyte activation, and myeloid leukocyte-mediated immunity were biological processes impacted by protein dysregulation in women destined to deliver within two weeks of diagnosis. The combination of AF protein abundance and quantitative cervical length improves prediction of the timing of delivery compared to cervical length alone, among women with a sonographic short cervix.
Similar content being viewed by others
Introduction
Preterm birth, the leading cause of perinatal morbidity and mortality1,2,3,4,5,6,7, is associated with an increased risk of short- and long-term health outcomes for neonates who survive8,9,10,11,12,13. Worldwide, preterm birth affects about 15 million babies annually, which accounts for the 11% global preterm birth rate3, 14, 15. In the United States, the rate of preterm birth has been approximately 10% since 2018, and this number remains high as compared to the rate observed in other developed countries3, 16, 17. The rate of preterm birth is even higher in several developing countries3, 4, 18 and contributes to substantial costs related to healthcare services19,20,21,22.
The identification of women at risk of preterm birth is central to the development of effective, preventive interventions aimed to reduce the potential negative effects at birth or later in life7, 23,24,25,26. Although various strategies to screen women at risk of preterm birth have been proposed by investigators, more accurate methods are yet to be developed, given the multifactorial causes leading to this syndrome7, 27, 28. Risk factors for preterm birth include advanced maternal age29, greater maternal body mass index30, 31, substance use during pregnancy (tobacco or alcohol use)32, 33, exposure to violence (physical or emotional)34,35,36, and psychosocial stress37, 38. In addition, clinical and obstetrical characteristics, such as a history of previous preterm birth39,40,41, gestational diabetes and chronic hypertension42, short inter-pregnancy interval43, 44, infection and inflammation45,46,47,48,49,50, genetic factors51,52,53,54, and environmental pollutants55,56,57,58,59, have also been linked to an increased risk of preterm birth. Race/ethnicity-related disparities in preterm birth rates were also reported60,61,62.
The traditional approach in the screening of imminent preterm birth involves a combination of maternal and obstetrical characteristics63,64,65. However, the detection rate of this approach is low (sensitivity ~ 20% and positive predictive value ~ 30%)63. Molecular biomarkers, such as fetal fibronectin in cervico-vaginal secretions66,67,68,69 and increased concentrations of interleukin (IL)-6 in amniotic fluid (AF)70,71,72,73 have also been associated with a higher risk of preterm birth. Novel proteomics platforms and bioinformatics algorithms have enabled a refined characterization of the AF proteome27, 74,75,76,77,78,79,80,81,82,83,84,85,86,87 for the prediction of several pathological conditions in pregnancy88,89,90, including preterm birth23, 91,92,93,94. For example, Lee et al.95 showed that AF cytokines and matrix metalloproteinases in combination with clinical risk factors improve the prediction of early preterm birth compared to a single protein or each clinical factor alone. Other investigators suggested a combination of multiple markers that include those profiled in AF and cervical fluid96,97,98.
In addition to biochemical markers, a powerful predictor of preterm birth is a transvaginal sonographic short cervix99,100,101,102,103,104,105,106, and women at risk can benefit from vaginal progesterone treatment107,108,109,110,111. We have proposed that the sensitivity of cervical length screening can be further improved by using a customized approach that accounts for maternal characteristics (weight, height, and parity) and exact gestational age at screening112. For women identified as at risk attributable to a short cervix at any time during the second or third trimester, it would be important, to know whether delivery is imminent. Therefore, in this study, we sought to identify AF proteins that can predict imminent preterm delivery in women with a sonographic short cervix.
Materials and methods
Study population and design
This was a retrospective analysis of data collected from pregnant women who were enrolled in a longitudinal biomarker study involving universal cervical length measurement at the Center for Advanced Obstetrical Care and Research of the Perinatology Research Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), the Detroit Medical Center, and Wayne State University (Detroit, MI, USA). Briefly, pregnant women were enrolled between 6 and 22 weeks of gestation and followed until delivery. Exclusion criteria included women who had the following diagnosis at the time of recruitment: preterm labor, preterm premature rupture of the membranes, preeclampsia, fetal growth restriction, active vaginal bleeding, multifetal gestation, and serious medical illness such as renal insufficiency, congestive heart disease, chronic respiratory insufficiency, etc. The protocol called for sonographic cervical length in the midtrimester followed by measurements every four weeks until 24 weeks of gestation, then every two weeks until delivery. When the cervical length measured was 25 mm or less, patients were sent to the obstetrical triage area for evaluation and counseling regarding risks of intra-amniotic infection/inflammation and preterm birth. Treatment with antibiotics were shown successful in a subset of patients with cervical insufficiency and intra-amniotic inflammation113, 114. The decision to offer amniocentesis was at the discretion of treating physicians.
The techniques for sonographic assessments of the short cervix and amniotic fluid sample collection were described in previous reports112, 115, 116. We retrospectively selected women with a singleton pregnancy and a sonographic short cervix (≤ 25 mm) between 16 and 32 weeks of gestation who had a transabdominal amniocentesis performed within two days of the cervical length measurement. Only cases without clinical signs of infection or labor at the time of amniocentesis were included. The primary indication for amniocentesis in this group of asymptomatic patients was to rule out intra-amniotic infection/inflammation due to a short cervix. For a subset of these patients, fetal karyotype and fetal lung maturity testing were also performed. Additional exclusion criteria for this study were labor induction for any reasons within two weeks of the amniocentesis, a positive AF culture for micro-organisms, abnormal fetal karyotypes or chromosomal microarray, and structural fetal anomalies. Participants in the study were recruited between May 2002 and September 2015, and all provided informed written consent prior to the collection of samples and images. The use of the data collected (demographic or clinical information, images, and samples) for research purposes was approved by the Human Investigation Committee of Wayne State University and the Institutional Review Board of NICHD. All methods were performed in accordance with relevant guidelines and regulations.
Proteomics profiling
The concentration of 1,310 proteins in AF samples was quantified by using the SOMAmer (Slow Off-rate Modified Aptamers) platform and its reagents, and proteomics profiling was performed by Somalogic, Inc. (Boulder, CO, USA), as described in previous publications117,118,119. Briefly, AF samples were diluted and then incubated with a mixture of SOMAmers on streptavidin-coated beads. Next, the beads were washed to remove all unbounded proteins and other matrix constituents, and proteins that remained bound to their cognate SOMAmer reagents were tagged with an NHS-biotin reagent. Pure cognate-SOMAmer complexes and unbound SOMAmer reagents were released from streptavidin beads by ultraviolet light that cleaved the photo-cleavable linker used to quantitate protein. The photo-cleavage eluate was separated from the beads and then incubated with a second streptavidin-coated bead. The free SOMAmer reagents were then removed during subsequent washing steps. In the final elution step, protein-bound SOMAmer reagents were released from their cognate proteins by using denaturing conditions. SOMAmer reagents were then quantified by hybridization to custom DNA microarrays. The cyanine-3 signal from the SOMAmer reagent was detected on the microarrays.
Statistical analyses
Demographic data analysis
The demographic and clinical characteristics were compared by using Wilcoxon-signed rank tests for continuous variables and Fisher’s exact tests for categorical variables. A p-value < 0.05 for the differences between the groups was considered statistically significant.
Proteomic data analysis
To identify AF protein dysregulation that can be informative about the timing of delivery, we fit linear models on log2-transformed relative fluorescence unit (RFU) values, using an explanatory variable for the delivery group: within two weeks (imminent delivery) vs. greater than two weeks until delivery. To account for the residual information that quantitative cervical length measurement may provide, we included cervical length as a covariate in the linear models. The significance of the group differences was assessed via moderated t-tests. An advantage of the moderate t-test, as contrasted with the standard t-test, is that it borrows information across the different proteins to derive more robust estimates of protein data variance120, and it has also been shown to improve the selection of predictors for omics data-based multi-variate predictive models121,122,123. Protein p-values were adjusted for multiple testing, and the false-positive discovery rate was controlled at the 10% level (q-value < 0.1). The linear models were fit by using the limma package in R/Bioconductor124.
Logistic regression models were also implemented to determine the odds of imminent delivery associated with a two-fold change in protein abundance, while adjusting for cervical length. The area under the receiver operating characteristic curve (AUC) was calculated to determine whether protein data improves the prediction of imminent delivery as compared to cervical length alone. Combinations of up to four proteins were also evaluated by using multivariate logistic regression and the AUC was determined. In addition, Kaplan–Meier survival curves based on the interval from amniocentesis to delivery were compared between patients with a risk score above and those with a risk score below a cut-off value corresponding to a 10% false-positive rate.
To identify biological processes overrepresented in the list of proteins associated with imminent delivery, we performed a Gene Ontology (GO) enrichment analysis with the clusterProfiler package125 in R/Bioconductor. The enrichment analyses also involved control for the false-positive discovery rate at 10% level. Visualization of the abundance of significant protein profiles was performed by using the heatmap function in the ComplexHeatmap package126.
Results
Demographic and clinical characteristics
The study included 90 women diagnosed with a sonographic short cervix (< 25 mm) during the second or third trimester. Of this group, 24 women delivered within two weeks from amniocentesis (n = 24) and the remaining 66 women delivered after two weeks from amniocentesis (n = 66). The characteristics of the study population are shown in Table 1, and the gestational ages at amniocentesis in both groups are depicted in Figure S1. The two groups were similar with respect to gestational age at amniocentesis, maternal age, weight, body mass index, race, parity, and history of preterm birth (p > 0.05). However, cervical length (median 5 vs. 15 mm, p < 0.001), gestational age at delivery (median 24.2 vs. 38.7 weeks, p < 0.001), and birthweight (median 651 vs. 2985 g, p < 0.001) were lower in women who delivered within two weeks compared to those who did not. In addition, neonates delivered within two weeks from amniocentesis had a significantly higher frequency of an Apgar score < 7 at 5 min [56.57% (13/23) vs. 7.7% (5/65), p < 0.001] and of admission to a neonatal intensive care unit [62.5% (15/24) vs. 13.6% (9/66), p < 0.001], compared to neonates whose delivery occurred after two weeks from amniocentesis. The presence of severe acute histologic chorioamnionitis was also more frequent among the women who delivered within two weeks of amniocentesis [81.2% (13/16) vs. 8.7% (4/46), p < 0.001]. Among the women who delivered within two weeks of amniocentesis, 50% (12/24) had intra-amniotic inflammation, indicated by an elevated AF concentration of IL-6 (IL-6 ≥ 2.6 ng/mL) compared to 7.6% (5/66) of those who delivered more than two weeks after an amniocentesis. Of note, the concentrations of IL-6 measured by ELISA were highly correlated to the relative fluorescence measures derived by the aptamer platform (Spearman’s correlation 0.89, p < 0.01).
Differential protein abundance predictive of preterm delivery within two weeks from amniocentesis
Of the 1,310 proteins profiled in AF, 17 were differentially abundant in women destined to deliver within two weeks of amniocentesis, independently of the cervical length (adjusted p-value < 0.10). A higher abundance of Synaptosome Associated Protein 25 (SNAP25) in AF was associated with lower odds of an earlier delivery (adjusted odds ratio [OR] = 0.39) (Fig. 1A). By contrast, the risk of preterm birth within two weeks of amniocentesis increased with the higher abundance of the following proteins: Glucose-6-Phosphate Isomerase (GPI), Protein Tyrosine Phosphatase Non-receptor type 11 (PTPN11), Oxidized Low-density Lipoprotein Receptor 1 (OLR1), Enolase 1 (ENO1), Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), Chitinase-3-like protein 1 (CHI3L1), Resistin (RETN), Colony-Stimulating Factor 3 (CSF3), Neutrophil gelatinase-associated lipocalin-2 (LCN2), C-X-C motif ligand 1 (CXCL1) and C-X-C motif ligand 8 (CXCL8), Peptidoglycan Recognition Protein 1 (PGLYRP1), Lactate Dehydrogenase B (LDHB), IL6, Matrix Metalloproteinase-8 (MMP8), and Proteinase 3 (PRTN3) (adjusted OR > 1.5). Of note, for all 17 proteins the significance p-value would be < 0.05 after adjusting for the secondary indication of amniocentesis, i.e. karyotype testing, which was slightly more frequent in women who delivered with two weeks (Table 1). This suggests that this confounding covariate was not a driver of the differential protein abundance observed herein. Therefore, we have attributed the proteomic differences observed to the pathophysiology leading to delivery within two weeks from amniocentesis.
Odds ratio (and 95% confidence intervals) for the association between amniotic fluid proteins and imminent delivery. (A) Odds ratios for delivery within two weeks and (B) within one week. Odds ratios are adjusted for quantitative cervical length. The odd-ratios are calculated for a two-fold change in protein abundance. Alpha-enolase (ENO1), C–C motif chemokine 2 (CCL2), C–C motif chemokine 4-like (CCL4L1), C–C motif chemokine 7 (CCL7), C-X-C motif ligand 8 (CXCL8), Carbohydrate sulfotransferase 15 (CHST15), Catalase (CAT), Chitinase-3-like protein 1 (CHI3L1), Collectin-12 (COLEC12), Glucose-6-phosphate isomerase (GPI), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Granulocyte colony-stimulating factor (CSF3), Gro-beta/gamma (CXCL3/CXCL2), Growth-regulated alpha protein (CXCL1), ICOS ligand (ICOSLG), Interleukin-6 (IL6), L-lactate dehydrogenase B chain (LDHB), Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), Matrix Metalloproteinase-8 (MMP8), Mitogen-activated protein kinase 14 (MAPK14), Myeloperoxidase (MPO), Neutrophil gelatinase-associated lipocalin (LCN2), Oxidized low-density lipoprotein receptor 1 (OLR1), Proteinase 3 (PRTN3), Peptidoglycan recognition protein 1 (PGLYRP1), Resistin (RETN), Small ubiquitin-related modifier 3 (SUMO3), SUMO-conjugating enzyme UBC9 (UBE2I), Synaptosomal-associated protein 25 (SNAP25), Tumor necrosis factor receptor superfamily member 1B (TNFRSF1B), Tyrosine-protein phosphatase non-receptor type 11 (PTPN11).
Differential protein abundance predictive of delivery within one week from amniocentesis
When comparing the protein abundance between women who delivered within one week from the amniocentesis (n = 9) to the group of women who delivered after one week (n = 81), we have identified 23 proteins with significant differential abundance after controlling the false discovery rate at the 10% level (q < 0.1). The cervical length adjusted ORs for the association of between a two-fold change in protein abundance and delivery within one week from amniocentesis are presented in Fig. 1B. Of note, among the 23 proteins that had higher abundance in the group of women who delivered within one week, nine were also identified as increased in women who delivered within two weeks from amniocentesis. Moreover, the point estimates of odds ratios for delivery with One week were larger than those for delivery within two weeks, suggesting a dose response relation between the timing of delivery and protein abundance changes.
Prediction of delivery within two weeks from amniocentesis by cervical length and amniotic fluid proteins
Although all women had an amniocentesis after diagnosis with a short cervix, the exact cervical length (quantitative assessment) was still predictive of delivery within two weeks from amniocentesis, and shorter cervical lengths were associated with increased risk (AUC = 0.74) (Fig. 2). The addition of data from one protein at a time led to improvements in the AUC that ranged from 4% to 12% depending on the specific protein (Fig. 2). The greatest improvement in the AUC statistic, compared to cervical length alone, was noted for GPI (AUC 0.86 vs 0.74), followed by CSF3, CXCL8, SNAP25, GAPDH, PGLYRP1, IL6, OLR1, and LDHB (p < 0.05 for all). Of note, the predictive value of the combination of cervical length and ELISA-based IL-6 (AUC = 0.8) was similar to that of cervical length and aptamers-based multiplex IL-6 (AUC = 0.83) (Figure S2).
ROC curves for predicting imminent delivery. Each panel compares prediction by cervical length alone and models that combine cervical length with protein abundance. AUC: area under the ROC curve and 95% confidence interval. CL: cervical length.
Combinations of the quantitative cervical length with up to four proteins further increased performance, reaching an AUC = 0.93 for the combination of CXCL8, MMP8, SNAP25, and PTPN11 (Fig. 3). The sensitivity, at a 10% false-positive rate, for the prediction of imminent delivery by the quantitative cervical length alone was 38%, yet it increased to 79% in combination with these four proteins. The Kaplan–Meier survival curves comparing duration to delivery between patients with a risk score above and those with a risk score below the risk cut-off value corresponding to a 10% false-positive rate are shown in Fig. 4. Patients with a risk score above the 10% false-positive rate cut-off value had a significantly shorter time to delivery compared to those with a risk score below the cut-off value [median: 1.4 (1.1–2.0) vs. 10.9 (9.8–13.3) weeks; log-rank p < 0.001].
Combinations of multiple proteins for improving prediction of imminent delivery. AUC: area under the ROC curve and 95% confidence interval. CL: cervical length.
Kaplan–Meier survival curves for patients with a risk score above and those with a risk score below the cut-off value corresponding to a 10% false positive rate. The risk scores are determined by quantitative cervical length and four proteins (CXCL8, SNAP25, PTPN11, MMP8).
Given the similar predictive value among several proteins, we applied cluster analysis and identified two main sets of proteins with higher abundance in pregnant women destined to deliver within two weeks of amniocentesis (Fig. 5). Cluster #1 was dominated by pro-inflammatory cytokines, including some previously associated with a high risk of preterm birth: MMP8, PGYRP1, PRTN3, CSF3, LCN2, RETN, IL6, CXCL8, CXCL1, and CHI3L1. Member proteins of cluster #2 were ENO1, GPI, OLR1, PTPN11, LDHB, and GAPDH. One protein, SNAP25, formed a cluster by itself, as it was negatively correlated with the risk of imminent delivery.
Clustered heatmap of protein data. The 17 proteins that were dysregulated in women destined for imminent delivery independent of cervical length were clustered using hierarchical clustering with correlation distance. Higher abundance of amniotic fluid protein levels is represented by a darker red color.
Gene ontology biological processes associated with imminent delivery
Enrichment analysis identified 23 biological processes associated with earlier preterm delivery after amniocentesis (q < 0.05, Table S1), which included neutrophil-mediated immunity, neutrophil activation, granulocyte activation, myeloid leukocyte activation, and myeloid leukocyte-mediated immunity (Fig. 6A,B).
Gene ontology enrichment analysis of amniotic fluid proteins dysregulated with imminent delivery. Top 20 enriched biological processes (A) and a network representation of proteins involved in the top five enriched biological processes (B). Protein Ratio represents the number of proteins significantly dysregulated per the total number of proteins in a particular biological process. Darker red/blue indicate high/low enrichment, respectively. The size of the dots corresponds to the protein ratio represented by each biological process.
Discussion
Principal findings of the study
(1) The AF proteins predicted imminent preterm delivery beyond what was previously possible when using only quantitative cervical length in women with a short cervix, a group already considered at risk for preterm birth (AUC = 0.93 for a combination of four proteins vs AUC = 0.74 for quantitative cervical length alone); (2) the sensitivity at a fixed false-positive rate of 10% for prediction of delivery within two weeks by a short cervix alone was 38%, yet it increased to 79% in combination with up to four AF proteins; and (3) neutrophil-mediated immunity, neutrophil activation, granulocyte activation, myeloid leukocyte activation, and myeloid leukocyte-mediated immunity were among the top biological processes associated with differentially abundant proteins in women who delivered within two weeks from an amniocentesis with a diagnosis of a short cervix.
Our findings in the context of what is already known
Herein, we have assessed the value of AF proteins for the prediction of imminent delivery in pregnant women with a sonographic short cervix. Typically defined as cervical length < 25 mm, a short cervix was shown to be associated with a higher risk of preterm birth than those with a long cervix at any time during preterm gestation. For the 8–31 weeks interval, the 25 mm cut-off value was more extreme (lower) than the 10th percentile among asymptomatic women with term delivery in this population112. Studies from our group have shown that the rate of intra-amniotic infection and inflammation are substantial among women with a short cervix115, 127, 128. Women with a short cervix are already at risk for preterm birth; hence, it is important for patient management to distinguish those destined for imminent delivery. For example, women at risk of delivery within one week from amniocentesis may benefit from the administration of antenatal steroids to improve fetal lung maturity. A unique feature of this study, which confers predictive value, is that the data had been collected prior to any eventual symptoms of preterm labor.
Previous studies have reported that an elevated concentration of IL-6 in the maternal circulation increases the risk for preterm birth94, 98, 129,130,131. Intra-amniotic inflammation, defined as IL-6 ≥ 2.6 ng/mL71, is a known risk factor for preterm labor and delivery70, 72, 91, 116, 132,133,134,135,136. Increased concentration of IL-6 in cervico-vaginal fluid has also been implicated in women who delivered preterm137, 138. Similarly, a sonographic short cervix is also a risk factor for preterm delivery116, 139. However, few studies have assessed the value of combining AF IL-6 concentrations with cervical length measurement for the prediction of spontaneous preterm birth97. In the current study, we demonstrated that the AF IL-6 concentration adds predictive value to the quantitative cervical length for the prediction of imminent preterm birth in asymptomatic pregnant women with a sonographic short cervix. The same finding holds for MMP8, which is in agreement with previous studies that have linked MMP8 to intra-amniotic infection/inflammation91, 135, 140,141,142,143,144, a causal pathway leading to preterm birth145,146,147,148,149,150,151,152,153,154,155,156.
Among the family of C-X-C motif chemokines, we observed a significant increase of CXCL1 and CXCL8 levels in patients destined to deliver within two weeks of amniocentesis. The most predictive of these proteins, CXCL8, is also known as IL-8157, 158. We have shown that an abundance of CXCL8 in AF combined with quantitative cervical length improves the prediction of preterm birth as compared to cervical length alone (AUC = 0.85 vs. 0.74, p = 0.022). Several prior studies have related an increased abundance of CXCL8 to an activation of the innate immune system in response to microbial infection/inflammation48, 50, 159,160,161, while others have argued that such elevation of CXCL8 is physiological as well, resulting from molecular changes in preparation for labor162,163,164,165. Of note, increased CXCL8 was also reported in cervico-vaginal fluid of women with preterm delivery166.
The use of multiple biomarkers seems imperative in the overall goal to improve the prediction of preterm birth, given the heterogeneity of these conditions and the multiple causal pathways95,96,97,98. In line with these studies, we combined quantitative cervical length with multiple AF proteins (CXCL8, MMP8, PTPN11, and SNAP25) and found a significant improvement in the AUC (AUC = 0.93 for the combined markers vs. AUC = 0.74 for cervical length alone, p = 0.006).
A possible role for neutrophil-mediated immunity in the intra-amniotic inflammatory response observed in pregnant women diagnosed with a short cervix
Neutrophils represent a primary cellular component of innate immunity that protects against microorganisms invading the amniotic cavity through an array of host defense mechanisms167, which may include phagocytosis168, the release of antimicrobial products and cytokines75, 169,170,171,172,173,174,175,176,177,178, and the formation of neutrophil extracellular traps178,179,180. Yet, neutrophils also form a physiological component of the AF cellular repertoire throughout pregnancy181; therefore, they are present in women diagnosed with a short cervix182. Herein, we found that biological processes, such as neutrophil-mediated immunity, neutrophil activation, granulocyte activation, myeloid leukocyte activation, and myeloid leukocyte-mediated immunity, were impacted by protein differential abundance in women who delivered within two weeks of the diagnosis of a short cervix. This finding suggests that AF neutrophils may undergo enhanced activation in women with a short cervix destined to deliver earlier preterm, either as a mechanism in response to bacterial products or “danger signal” in cases of sterile intra-amniotic inflammation115. However, further investigation is required to elucidate the participation of AF neutrophils in the inflammatory processes leading to earlier preterm delivery in women with a short cervix.
Strength and limitations
This is the first study providing a comprehensive evaluation of AF proteins for the prediction of imminent delivery among asymptomatic pregnant women diagnosed with a sonographic short cervix. Some of the AF proteins we identified in the present study (GPI, PTRN11, OLR1, ENO1, GAPDH, CHI3L1, CSF3, LCN2, PGLYRP1, LDHB, PRTN3, and SNAP25) have not been widely explored in previous studies; therefore, they could provide additional insight into the discovery of biomarkers for further understanding of the pathophysiologic pathways leading to preterm birth23. Furthermore, the results of this study contribute to the growing interest in the use of multiple markers to predict preterm birth183. Our study demonstrates that when an asymptomatic patient presents with a sonographic short cervix between 16 and 32 weeks of gestation, specific AF proteins provide additional predictive power for identifying women at risk of imminent delivery (e.g. within 1 or 2 weeks), relative to cervical length alone. Limitations of this study are attributable to the timing of cervical length assessment and amniocentesis being within two days of each other, the limited power for assessing multi-variate prediction of delivery within one week of amniocentesis, and missing detailed obstetrical history such as type of prior preterm term birth and cervical surgery.
Conclusions
Amniotic fluid protein abundance is predictive of imminent delivery among asymptomatic women with a sonographic short cervix. The combination of AF proteins and quantitative cervical length measurement provides improved prediction of the timing of delivery compared to cervical length measurement alone, and this finding could have implications for patient management.
References
Goldenberg, R. et al. Epidemiology and causes of preterm birth. Lancet 371(9606), 75–84 (2008).
Liu, L. et al. Global, regional, and national causes of child mortality in 2000–13, with projections to inform post-2015 priorities: An updated systematic analysis. Lancet 385(9966), 430–440 (2015).
Blencowe, H. et al. National, regional, and worldwide estimates of preterm birth rates in the year 2010 with time trends since 1990 for selected countries: A systematic analysis and implications. Lancet 379, 2162–2172 (2012).
Chawanpaiboon, S. et al. Global, regional, and national estimates of levels of preterm birth in 2014: A systematic review and modelling analysis. Lancet Glob. Health 7(1), e37–e46 (2019).
Jones, E. O., Liew, Z. Q. & Rust, O. A. The short cervix: A critical analysis of diagnosis and treatment. Obstet. Gynecol. Clin. North Am. 47(4), 545–567 (2020).
Iams, J. D. & Berghella, V. Care for women with prior preterm birth. Am. J. Obstet. Gynecol. 203(2), 89–100 (2010).
Romero, R., Dey, S. K. & Fisher, S. J. Preterm labor: One syndrome, many causes. Science 345(6198), 760–765 (2014).
Saigal, S. & Doyle, L. W. An overview of mortality and sequelae of preterm birth from infancy to adulthood. Lancet 371(9608), 261–269 (2008).
Dong, Y. & Yu, J. L. An overview of morbidity, mortality and long-term outcome of late preterm birth. World J. Pediatr. 7(3), 199–204 (2011).
Mwaniki, M. K. et al. Long-term neurodevelopmental outcomes after intrauterine and neonatal insults: A systematic review. Lancet 379(9814), 445–452 (2012).
Luu, T. M., Rehman Mian, M. O. & Nuyt, A. M. Long-term impact of preterm birth: Neurodevelopmental and physical health outcomes. Clin. Perinatol. 44(2), 305–314 (2017).
Chehade, H. et al. Preterm birth: Long term cardiovascular and renal consequences. Curr. Pediatr. Rev. 14(4), 219–226 (2018).
Tamm, L. et al. Early brain abnormalities in infants born very preterm predict under-reactive temperament. Early Hum. Dev. 144, 104985 (2020).
Liu, L. et al. Global, regional, and national causes of under-5 mortality in 2000–15: An updated systematic analysis with implications for the Sustainable Development Goals. Lancet 388(10063), 3027–3035 (2016).
Walani, S. R. Global burden of preterm birth. Int. J. Gynaecol. Obstet. 150(1), 31–33 (2020).
Bronstein, J. M., Wingate, M. S. & Brisendine, A. E. Why is the U.S. preterm birth rate so much higher than the rates in Canada, Great Britain, and Western Europe?. Int. J. Health Serv. 48(4), 622–640 (2018).
Martin, J.A., et al., Births: Final data for 2019. National Center for Health Statistics, 2021.
Wagura, P. et al. Prevalence and factors associated with preterm birth at Kenyatta national hospital. BMC Pregnancy Childbirth 18(1), 107 (2018).
Petrou, S. Economic consequences of preterm birth and low birthweight. BJOG 110(20), 17–23 (2003).
Underwood, M. A., Danielsen, B. & Gilbert, W. M. Cost, causes and rates of rehospitalization of preterm infants. J. Perinatol. 27(10), 614–619 (2007).
Hodek, J. M., von der Schulenburg, J. M. & Mittendorf, T. Measuring economic consequences of preterm birth—Methodological recommendations for the evaluation of personal burden on children and their caregivers. Health Econ. Rev. 1(1), 6 (2011).
Lakshmanan, A. et al. The impact of preterm birth <37 weeks on parents and families: a cross-sectional study in the 2 years after discharge from the neonatal intensive care unit. Health Qual. Life Outcomes 15(1), 38 (2017).
Goldenberg, R. L., Goepfert, A. R. & Ramsey, P. S. Biochemical markers for the prediction of preterm birth. Am. J. Obstet. Gynecol. 192(5 Suppl), S36-46 (2005).
Chang, H. H. et al. Preventing preterm births: analysis of trends and potential reductions with interventions in 39 countries with very high human development index. Lancet 381(9862), 223–234 (2013).
Lackritz, E. M. et al. A solution pathway for preterm birth: Accelerating a priority research agenda. Lancet Glob. Health 1(6), e328–e330 (2013).
Newnham, J. P. et al. Reducing preterm birth by a statewide multifaceted program: An implementation study. Am. J. Obstet. Gynecol. 216(5), 434–442 (2017).
Romero, R. et al. The use of high-dimensional biology (genomics, transcriptomics, proteomics, and metabolomics) to understand the preterm parturition syndrome. BJOG 113(Suppl 3), 118–135 (2006).
Sheikh, I. A. et al. Spontaneous preterm birth and single nucleotide gene polymorphisms: a recent update. BMC Genomics 17(Suppl 9), 759 (2016).
Delbaere, I. et al. Pregnancy outcome in primiparae of advanced maternal age. Eur. J. Obstet. Gynecol. Reprod. Biol. 135(1), 41–46 (2007).
Torloni, M. R. et al. Maternal BMI and preterm birth: A systematic review of the literature with meta-analysis. J. Matern. Fetal Neonatal. Med. 22(11), 957–970 (2009).
Slack, E. et al. Maternal obesity classes, preterm and post-term birth: A retrospective analysis of 479,864 births in England. BMC Pregnancy Childbirth 19(1), 434 (2019).
Shiono, P. H., Klebanoff, M. A. & Rhoads, G. G. Smoking and drinking during pregnancy their effects on preterm birth. JAMA 255, 82–84 (1986).
Soneji, S. & Beltran-Sanchez, H. Association of maternal cigarette smoking and smoking cessation with preterm birth. JAMA Netw. Open 2(4), e192514 (2019).
Sanchez, S. E. et al. Risk of spontaneous preterm birth in relation to maternal exposure to intimate partner violence during pregnancy in Peru. Matern. Child Health J. 17(3), 485–492 (2013).
Silverman, J. G. et al. Intimate partner violence victimization prior to and during pregnancy among women residing in 26 US states: Associations with maternal and neonatal health. Am. J. Obstet. Gynecol. 195(1), 140–148 (2006).
Sigalla, G. N. et al. Intimate partner violence during pregnancy and its association with preterm birth and low birth weight in Tanzania: A prospective cohort study. PLoS ONE 12(2), e0172540 (2017).
McDonald, S. W. et al. Cumulative psychosocial stress, coping resources, and preterm birth. Arch. Womens Ment. Health 17(6), 559–568 (2014).
Tanpradit, K. & Kaewkiattikun, K. The effect of perceived stress during pregnancy on preterm birth. Int. J. Womens Health 12, 287–293 (2020).
Mazaki-Tovi, S. et al. Recurrent preterm birth. Semin. Perinatol. 31(3), 142–158 (2007).
Laughon, S. K. et al. The NICHD Consecutive Pregnancies Study: Recurrent preterm delivery by subtype. Am. J. Obstet. Gynecol. 210(2), 131 e1–8 (2014).
Yang, J. et al. Recurrence of preterm birth and early term birth. Obstet. Gynecol. 128(2), 364–372 (2016).
Berger, H. et al. Impact of diabetes, obesity and hypertension on preterm birth: Population-based study. PLoS ONE 15(3), e0228743 (2020).
de Weger, F. J. et al. Advanced maternal age, short interpregnancy interval, and perinatal outcome. Am. J. Obstet. Gynecol. 204(5), 421 e1–9 (2011).
Schummers, L. et al. Association of short interpregnancy interval with pregnancy outcomes according to maternal age. JAMA Intern. Med. 178(12), 1661–1670 (2018).
Gibbs, R. S. et al. A review of premature birth and subclinical infection. Am. J. Obstet. Gynecol. 166(5), 1515–1528 (1992).
Romero, R. et al. Infection and prematurity and the role of preventive strategies. Semin. Neonatol. 7(4), 259–274 (2002).
Romero, R. et al. Bacterial vaginosis, the inflammatory response and the risk of preterm birth: A role for genetic epidemiology in the prevention of preterm birth. Am. J. Obstet. Gynecol. 190(6), 1509–1519 (2004).
Agrawal, V. & Hirsch, E. Intrauterine infection and preterm labor. Semin. Fetal Neonatal. Med. 17(1), 12–19 (2012).
Verma, I., Avasthi, K. & Berry, V. Urogenital infections as a risk factor for preterm labor: A hospital-based case-control study. J. Obstet. Gynaecol. India 64(4), 274–278 (2014).
Romero, R. et al. The role of inflammation and infection in preterm birth. Semin. Reprod. Med. 25(1), 21–39 (2007).
York, T. P. et al. Racial differences in genetic and environmental risk to preterm birth. PLoS ONE 5(8), e12391 (2010).
Dolan, S. M. et al. Synopsis of preterm birth genetic association studies: the preterm birth genetics knowledge base (PTBGene). Public Health Genomics 13(7–8), 514–523 (2010).
Esplin, M. S. et al. Cluster analysis of spontaneous preterm birth phenotypes identifies potential associations among preterm birth mechanisms. Am. J. Obstet. Gynecol. 213(3), 429 e1–9 (2015).
Strauss, J. F. 3rd. et al. Spontaneous preterm birth: Advances toward the discovery of genetic predisposition. Am. J. Obstet. Gynecol. 218(3), 294-314 e2 (2018).
Lin, Y. T. et al. Associations between ozone and preterm birth in women who develop gestational diabetes. Am. J. Epidemiol. 181(4), 280–287 (2015).
Li, X. et al. Association between ambient fine particulate matter and preterm birth or term low birth weight: An updated systematic review and meta-analysis. Environ. Pollut. 227, 596–605 (2017).
Padula, A. M. et al. Environmental pollution and social factors as contributors to preterm birth in Fresno County. Environ. Health 17(1), 70 (2018).
Huang, H. et al. Investigation of association between environmental and socioeconomic factors and preterm birth in California. Environ. Int. 121(Pt 2), 1066–1078 (2018).
Ju, L. et al. Maternal air pollution exposure increases the risk of preterm birth: Evidence from the meta-analysis of cohort studies. Environ. Res. 202, 111654 (2021).
Culhane, J. F. & Goldenberg, R. L. Racial disparities in preterm birth. Semin. Perinatol. 35(4), 234–239 (2011).
Wallace, M. E. et al. Racial/ethnic differences in preterm perinatal outcomes. Am. J. Obstet. Gynecol. 216(3), 306 e1-306 e12 (2017).
Purisch, S. E. & Gyamfi-Bannerman, C. Epidemiology of preterm birth. Semin. Perinatol. 41(7), 387–391 (2017).
Mercer, B. M. et al. The preterm prediction study: A clinical risk assessment system. Am. J. Obstet. Gynecol. 174, 1885–1895 (1996).
Beta, J. et al. Prediction of spontaneous preterm delivery from maternal factors, obstetric history and placental perfusion and function at 11–13 weeks. Prenat. Diagn. 31(1), 75–83 (2011).
Fuchs, F. et al. Predictive score for early preterm birth in decisions about emergency cervical cerclage in singleton pregnancies. Acta Obstet. Gynecol. Scand. 91(6), 744–749 (2012).
Peaeeman, A. M. et al. Fetal fibronectin as a predictor of preterm birth with symptoms: A multicenter trial. Am. J. Obstet. Gynecol. 177, 13–18 (1997).
Chien, P. F. et al. The diagnostic accuracy of cervico-vaginal fetal fibronectin in predicting preterm delivery: an overview. BJOG 104, 436–444 (1997).
Leitich, H. et al. Cervicovaginal foetal fibronectin as a marker for preterm delivery: A meta-analysis. Am. J. Obstet. Gynecol. 180, 1169–1176 (1999).
Hezelgrave, N. L. & Shennan, A. H. Quantitative fetal fibronectin to predict spontaneous preterm birth: A review. Womens Health (Lond.) 12, 121–128 (2016).
Wenstrom, K. D. et al. Elevated second-trimester amniotic fluid interleukin-6 levels predict preterm delivery. Am. J. Obstet. Gynecol. 178, 546–550 (1998).
Yoon, B. H. et al. Clinical significance of intra-amniotic inflammation in patients with preterm labor and intact membranes. Am. J. Obstet. Gynecol. 185(5), 1130–1136 (2001).
Gervasi, M. T. et al. Midtrimester amniotic fluid concentrations of interleukin-6 and interferon-gamma-inducible protein-10: Evidence for heterogeneity of intra-amniotic inflammation and associations with spontaneous early (<32 weeks) and late (>32 weeks) preterm delivery. J. Perinat. Med. 40(4), 329–343 (2012).
Leanos-Miranda, A. et al. Interleukin-6 in amniotic fluid: A reliable marker for adverse outcomes in women in preterm labor and intact membranes. Fetal Diagn. Ther. 48(4), 313–320 (2021).
Vuadens, F. et al. Identification of biologic markers of the premature rupture of fetal membranes: Proteomic approach. Proteomics 3(8), 1521–1525 (2003).
Gravett, M. G. et al. Diagnosis of intraamniotic infection by proteomic profiling and identification of novel biomarkers. JAMA 292(4), 462–469 (2004).
Buhimschi, I. A., Christner, R. & Buhimschi, C. S. Proteomic biomarker analysis of amniotic fluid for identification of intra-amniotic inflammation. BJOG 112(2), 173–181 (2005).
Michaels, J. E. et al. Comprehensive proteomic analysis of the human amniotic fluid proteome: Gestational age-dependent changes. J. Proteome Res. 6(4), 1277–1285 (2007).
Queloz, P. A. et al. Proteomic analyses of amniotic fluid: Potential applications in health and diseases. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 850(1–2), 336–342 (2007).
Bujold, E. et al. Proteomic profiling of amniotic fluid in preterm labor using two-dimensional liquid separation and mass spectrometry. J. Matern. Fetal Neonatal. Med. 21(10), 697–713 (2008).
Buhimschi, I. A. et al. Multidimensional proteomics analysis of amniotic fluid to provide insight into the mechanisms of idiopathic preterm birth. PLoS ONE 3(4), e2049 (2008).
Romero, R. et al. Proteomic analysis of amniotic fluid to identify women with preterm labor and intra-amniotic inflammation/infection: The use of a novel computational method to analyze mass spectrometric profiling. J. Matern. Fetal Neonatal. Med. 21(6), 367–388 (2008).
Fotopoulou, C. et al. Proteomic analysis of midtrimester amniotic fluid to identify novel biomarkers for preterm delivery. J. Matern. Fetal Neonatal. Med. 25(12), 2488–2493 (2012).
Tambor, V. et al. Proteomics and bioinformatics analysis reveal underlying pathways of infection associated histologic chorioamnionitis in pPROM. Placenta 34(2), 155–161 (2013).
Bahado-Singh, R. O. et al. Artificial intelligence and amniotic fluid multiomics: Prediction of perinatal outcome in asymptomatic women with short cervix. Ultrasound Obstet. Gynecol. 54(1), 110–118 (2019).
Hong, S. et al. Identifying potential biomarkers related to pre-term delivery by proteomic analysis of amniotic fluid. Sci. Rep. 10(1), 19648 (2020).
Jeon, H. S. et al. Proteomic biomarkers in mid-trimester amniotic fluid associated with adverse pregnancy outcomes in patients with systemic lupus erythematosus. PLoS ONE 15(7), e0235838 (2020).
Bhatti, G. et al. The amniotic fluid cell-free transcriptome in spontaneous preterm labor. Sci. Rep. 11(1), 13481 (2021).
Cho, C. K. et al. Proteomics analysis of human amniotic fluid. Mol. Cell. Proteomics 6, 1406–1415 (2007).
Tsangaris, G. T. et al. Application of proteomics for the identification of biomarkers in amniotic fluid: Are we ready to provide a reliable prediction?. EPMA J. 2(2), 149–155 (2011).
Kamath-Rayne, B. D. et al. Amniotic fluid: The use of high-dimensional biology to understand fetal well-being. Reprod. Sci. 21(1), 6–19 (2014).
Lee, S. M. et al. Mid-trimester amniotic fluid pro-inflammatory biomarkers predict the risk of spontaneous preterm delivery in twins: A retrospective cohort study. J. Perinatol. 35(8), 542–546 (2015).
Hallingstrom, M. et al. Mid-trimester amniotic fluid proteome’s association with spontaneous preterm delivery and gestational duration. PLoS ONE 15(5), e0232553 (2020).
Hsu, T. Y. et al. Identifying the potential protein biomarkers of preterm birth in amniotic fluid. Taiwan J. Obstet. Gynecol. 59(3), 366–371 (2020).
Tarca, A. L. et al. Crowdsourcing assessment of maternal blood multi-omics for predicting gestational age and preterm birth. Cell Rep. Med. 2(6), 100323 (2021).
Lee, S. M. et al. Prediction of spontaneous preterm birth in women with cervical insufficiency: Comprehensive analysis of multiple proteins in amniotic fluid. J. Obstet. Gynaecol. Res. 42(7), 776–783 (2016).
Goldenberg, R. L. et al. The Preterm Prediction Study: Toward a multiple-marker test for spontaneous preterm birth. Am. J. Obstet. Gynecol. 185, 643–651 (2001).
Holst, R. M. et al. Prediction of spontaneous preterm delivery in women with preterm labor: Analysis of multiple proteins in amniotic and cervical fluids. Obstet. Gynecol. 114, 268–277 (2009).
Huang, L. et al. Serum multiple cytokines for the prediction of spontaneous preterm birth in asymptomatic women: A nested case-control study. Cytokine 117, 91–97 (2019).
Iams, J. D. 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. 334(9), 567–572 (1996).
Hassan, S. S. et al. Patients with an ultrasonographic cervical length < or =15 mm have nearly a 50% risk of early spontaneous preterm delivery. Am. J. Obstet. Gynecol. 182(6), 1458–1467 (2000).
Romero, R. et al. A blueprint for the prevention of preterm birth: Vaginal progesterone in women with a short cervix. J. Perinat. Med. 41(1), 27–44 (2013).
Hiersch, L. et al. Role of cervical length measurement for preterm delivery prediction in women with threatened preterm labor and cervical dilatation. J. Ultrasound Med. 35(12), 2631–2640 (2016).
Son, M. & Miller, E. S. Predicting preterm birth: Cervical length and fetal fibronectin. Semin. Perinatol. 41(8), 445–451 (2017).
Berghella, V. et al. Cerclage for sonographic short cervix in singleton gestations without prior spontaneous preterm birth: Systematic review and meta-analysis of randomized controlled trials using individual patient-level data. Ultrasound Obstet. Gynecol. 50(5), 569–577 (2017).
Rosenbloom, J. I. et al. Predictive value of midtrimester universal cervical length screening based on parity. J. Ultrasound Med. 39(1), 147–154 (2020).
Maia, M. C. et al. Is cervical length evaluated by transvaginal ultrasonography helpful in detecting true preterm labor?. J. Matern. Fetal Neonatal. Med. 33(17), 2902–2908 (2020).
Fonseca, E. B. et al. Progesterone and the risk of preterm birth among women with a short cervix. N. Engl. J. Med. 357(5), 462–469 (2007).
Hassan, S. S. 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. 38(1), 18–31 (2011).
Romero, R. et al. Vaginal progesterone decreases preterm birth and neonatal morbidity and mortality in women with a twin gestation and a short cervix: An updated meta-analysis of individual patient data. Ultrasound Obstet. Gynecol. 49(3), 303–314 (2017).
Romero, R. et al. Vaginal progesterone for preventing preterm birth and adverse perinatal outcomes in singleton gestations with a short cervix: A meta-analysis of individual patient data. Am. J. Obstet. Gynecol. 218(2), 161–180 (2018).
Conde-Agudelo, A. et al. Vaginal progesterone is as effective as cervical cerclage to prevent preterm birth in women with a singleton gestation, previous spontaneous preterm birth, and a short cervix: Updated indirect comparison meta-analysis. Am. J. Obstet. Gynecol. 219(1), 10–25 (2018).
Gudicha, D. W. et al. Personalized assessment of cervical length improves prediction of spontaneous preterm birth: A standard and a percentile calculator. Am. J. Obstet. Gynecol. 224(3), 288 e1-288 e17 (2021).
Oh, K. J. et al. Evidence that antibiotic administration is effective in the treatment of a subset of patients with intra-amniotic infection/inflammation presenting with cervical insufficiency. Am. J. Obstet. Gynecol. 221(2), 140 e1-140 e18 (2019).
Yeo, L., et al., Resolution of acute cervical insufficiency after antibiotics in a case with amniotic fluid sludge. J. Matern. Fetal Neonatal. Med. 1–11 (2021).
Romero, R. et al. Sterile intra-amniotic inflammation in asymptomatic patients with a sonographic short cervix: Prevalence and clinical significance. J. Matern. Fetal Neonatal. Med. 28(11), 1343–1359 (2015).
Tarca, A.L., et al., The cytokine network in women with an asymptomatic short cervix and the risk of preterm delivery. Am. J. Reprod. Immunol. 78(3) (2017).
Gold, L. et al. Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS ONE 5(12), e15004 (2010).
Davies, D. R. et al. Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets. Proc. Natl. Acad. Sci. USA 109(49), 19971–19976 (2012).
Romero, R. et al. The maternal plasma proteome changes as a function of gestational age in normal pregnancy: A longitudinal study. Am. J. Obstet. Gynecol. 217(1), 671–6721 (2017).
Phipson, B. et al. Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression. Ann. Appl. Stat. 10(2), 946–963 (2016).
Tarca, A. L. et al. Strengths and limitations of microarray-based phenotype prediction: Lessons learned from the IMPROVER diagnostic signature challenge. Bioinformatics 29(22), 2892–2899 (2013).
Belcastro, V. et al. The sbv IMPROVER systems toxicology computational challenge: Identification of human and species-independent blood response markers as predictors of smoking exposure and cessation status. Comput. Toxicol. 5, 38–51 (2018).
Dayarian, A. et al. Predicting protein phosphorylation from gene expression: Top methods from the IMPROVER species translation challenge. Bioinformatics 31(4), 462–470 (2015).
Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43(7), e47 (2015).
Yu, G. et al. clusterProfiler: An R package for comparing biological themes among gene clusters. OMICS 16(5), 284–287 (2012).
Gu, Z., Eils, R. & Schlesner, M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32(18), 2847–2849 (2016).
Vaisbuch, E. et al. Patients with an asymptomatic short cervix (<or=15 mm) have a high rate of subclinical intraamniotic inflammation: Implications for patient counseling. Am. J. Obstet. Gynecol. 202(5), 4331–4338 (2010).
Hassan, S. et al. A sonographic short cervix as the only clinical manifestation of intra-amniotic infection. J. Perinat. Med. 34(1), 13–19 (2006).
Kramer, M. S. et al. Mid-trimester maternal plasma cytokines and CRP as predictors of spontaneous preterm birth. Cytokine 49(1), 10–14 (2010).
Sorokin, Y. et al. Maternal serum interleukin-6, C-reactive protein, and matrix metalloproteinase-9 concentrations as risk factors for preterm birth <32 weeks and adverse neonatal outcomes. Am. J. Perinatol. 27(8), 631–640 (2010).
Shahshahan, Z. & Hashemi, L. Maternal serum cytokines in the prediction of preterm labor and response to tocolytic therapy in preterm labor women. Adv. Biomed. Res. 3, 126 (2014).
Figueroa, R. et al. Evaluation of amniotic fluid cytokines in preterm labor and intact membranes. J. Matern. Fetal Neonatal. Med. 18(4), 241–247 (2005).
Thomakos, N. et al. Amniotic fluid interleukin-6 and tumor necrosis factor-alpha at mid-trimester genetic amniocentesis: Relationship to intra-amniotic microbial invasion and preterm delivery. Eur. J. Obstet. Gynecol. Reprod. Biol. 148(2), 147–151 (2010).
La Sala, G. B. et al. Protein microarrays on midtrimester amniotic fluids: A novel approach for the diagnosis of early intrauterine inflammation related to preterm delivery. Int. J. Immunopathol. Pharmacol. 25, 1029–1040 (2012).
Kim, A. et al. Identification of biomarkers for preterm delivery in mid-trimester amniotic fluid. Placenta 34(10), 873–878 (2013).
Theis, K. R. et al. Microbial burden and inflammasome activation in amniotic fluid of patients with preterm prelabor rupture of membranes. J. Perinat. Med. 48(2), 115–131 (2020).
Coleman, M. A. et al. Predicting preterm delivery: Comparison of cervicovaginal interleukin (IL)-1beta, IL-6 and IL-8 with fetal fibronectin and cervical dilatation. Eur. J. Obstet. Gynecol. Reprod. Biol. 95(2), 154–158 (2001).
Torbe, A. & Czajka, R. Proinflammatory cytokines and other indications of inflammation in cervico-vaginal secretions and preterm delivery. Int. J. Gynaecol. Obstet. 87(2), 125–130 (2004).
Kiefer, D. G. et al. Amniotic fluid inflammatory score is associated with pregnancy outcome in patients with mid trimester short cervix. Am. J. Obstet. Gynecol. 206(1), 68 e1–6 (2012).
Weiss, A., Goldman, S. & Shalev, E. The matrix metalloproteinases (MMPS) in the decidua and fetal membranes. Front Biosci 12, 649–659 (2007).
Park, C. W. et al. The antenatal identification of funisitis with a rapid MMP-8 bedside test. J. Perinat. Med. 36(6), 497–502 (2008).
Oh, K. J. et al. Detection of ureaplasmas by the polymerase chain reaction in the amniotic fluid of patients with cervical insufficiency. J. Perinat. Med. 38(3), 261–268 (2010).
Park, C. W. et al. The frequency and clinical significance of intra-amniotic inflammation defined as an elevated amniotic fluid matrix metalloproteinase-8 in patients with preterm labor and low amniotic fluid white blood cell counts. Obstet. Gynecol. Sci. 56(3), 167–175 (2013).
Kim, S. M. et al. The relationship between the intensity of intra-amniotic inflammation and the presence and severity of acute histologic chorioamnionitis in preterm gestation. J. Matern. Fetal Neonatal. Med. 28(13), 1500–1509 (2015).
Gravett, M. G. et al. An experimental model for intraamniotic infection and preterm labor in rhesus monkeys. Am. J. Obstet. Gynecol. 171(6), 1660–1667 (1994).
Gravett, M. G. et al. Immunomodulators plus antibiotics delay preterm delivery after experimental intraamniotic infection in a nonhuman primate model. Am. J. Obstet. Gynecol. 197(5), 518 e1–8 (2007).
Novy, M. J. et al. Ureaplasma parvum or Mycoplasma hominis as sole pathogens cause chorioamnionitis, preterm delivery, and fetal pneumonia in rhesus macaques. Reprod Sci 16(1), 56–70 (2009).
Grigsby, P. L. et al. Maternal azithromycin therapy for Ureaplasma intraamniotic infection delays preterm delivery and reduces fetal lung injury in a primate model. Am. J. Obstet. Gynecol. 207(6), 475 e1-475 e14 (2012).
Gomez-Lopez, N. et al. Intra-amniotic administration of HMGB1 induces spontaneous preterm labor and birth. Am. J. Reprod. Immunol. 75(1), 3–7 (2016).
Gomez-Lopez, N. et al. Intra-amniotic administration of lipopolysaccharide induces spontaneous preterm labor and birth in the absence of a body temperature change. J. Matern. Fetal Neonatal. Med. 31(4), 439–446 (2018).
Gomez-Lopez, N. et al. Inhibition of the NLRP3 inflammasome can prevent sterile intra-amniotic inflammation, preterm labor/birth, and adverse neonatal outcomes. Biol. Reprod. 100(5), 1306–1318 (2019).
Faro, J. et al. Intra-amniotic inflammation induces preterm birth by activating the NLRP3 inflammasomedagger. Biol. Reprod. 100(5), 1290–1305 (2019).
Coleman, M. et al. A broad spectrum chemokine inhibitor prevents preterm labor but not microbial invasion of the amniotic cavity or neonatal morbidity in a non-human primate model. Front. Immunol. 11, 770 (2020).
Motomura, K., et al., Intra-amniotic infection with Ureaplasma parvum causes preterm birth and neonatal mortality that are prevented by treatment with clarithromycin. mBio 11(3) (2020).
Motomura, K., et al., The alarmin interleukin-1alpha causes preterm birth through the NLRP3 inflammasome. Mol. Hum. Reprod. (2020).
Galaz, J. et al. Betamethasone as a potential treatment for preterm birth associated with sterile intra-amniotic inflammation: A murine study. J. Perinat. Med. 49(7), 897–906 (2021).
Brat, D. J., Bellail, A. C. & Van Meir, E. G. The role of Interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis. Neuro-oncol. 7, 122–133 (2005).
Russo, R. C. et al. The CXCL8/IL-8 chemokine family and its receptors in inflammatory diseases. Expert. Rev. Clin. Immunol. 10(5), 593–619 (2014).
Witt, A. et al. IL-8 concentrations in maternal serum, amniotic fluid and cord blood in relation to different pathogens within the amniotic cavity. J. Perinat. Med. 33(1), 22–26 (2005).
Romero, R. et al. The preterm parturition syndrome. BJOG 113(Suppl 3), 17–42 (2006).
Romero, R. et al. Evidence of perturbations of the cytokine network in preterm labor. Am. J. Obstet. Gynecol. 213(6), 8361–83618 (2015).
Saito, S. et al. Detection and localization of interleukin-8 mRNA and protein in human placenta and decidual tissues. J. Reprod. Immunol. 27, 161–172 (1994).
Gomez-Lopez, N. et al. The role of chemokines in term and premature rupture of the fetal membranes: A review. Biol. Reprod. 82(5), 809–814 (2010).
Hamilton, S. A., Tower, C. L. & Jones, R. L. Identification of chemokines associated with the recruitment of decidual leukocytes in human labour: potential novel targets for preterm labour. PLoS ONE 8(2), e56946 (2013).
Gomez-Lopez, N. et al. Immune cells in term and preterm labor. Cell Mol. Immunol. 11(6), 571–581 (2014).
Kedzierska-Markowicz, A. et al. Evaluation of the correlation between IL-1beta, IL-8, IFN-gamma cytokine concentration in cervico-vaginal fluid and the risk of preterm delivery. Ginekol. Pol. 86(11), 821–826 (2015).
Gomez-Lopez, N. et al. RNA sequencing reveals diverse functions of amniotic fluid neutrophils and monocytes/macrophages in intra-amniotic infection. J. Innate Immun. 13(2), 63–82 (2021).
Gomez-Lopez, N., et al., Amniotic fluid neutrophils can phagocytize bacteria: A mechanism for microbial killing in the amniotic cavity. Am. J. Reprod. Immunol. 78(4) (2017).
Heller, K. A., Greig, P. C. & Heine, R. P. Amniotic-fluid lactoferrin: A marker for subclinical intraamniotic infection prior to 32 weeks gestation. Infect. Dis. Obstet. Gynecol. 3(5), 179–183 (1995).
Otsuki, K. et al. Amniotic fluid lactoferrin in intrauterine infection. Placenta 20(2–3), 175–179 (1999).
Pacora, P. et al. Lactoferrin in intrauterine infection, human parturition, and rupture of fetal membranes. Am. J. Obstet. Gynecol. 183(4), 904–910 (2000).
Maymon, E. et al. Value of amniotic fluid neutrophil collagenase concentrations in preterm premature rupture of membranes. Am. J. Obstet. Gynecol. 185(5), 1143–1148 (2001).
Espinoza, J. et al. Antimicrobial peptides in amniotic fluid: Defensins, calprotectin and bacterial/permeability-increasing protein in patients with microbial invasion of the amniotic cavity, intra-amniotic inflammation, preterm labor and premature rupture of membranes. J. Matern. Fetal Neonatal. Med. 13(1), 2–21 (2003).
Soto, E. et al. Human beta-defensin-2: A natural antimicrobial peptide present in amniotic fluid participates in the host response to microbial invasion of the amniotic cavity. J. Matern. Fetal. Neonatal. Med. 20(1), 15–22 (2007).
Martinez-Varea, A. et al. Clinical chorioamnionitis at term VII: The amniotic fluid cellular immune response. J. Perinat. Med. 45(5), 523–538 (2017).
Varrey, A. et al. Human β-defensin-1: A natural antimicrobial peptide present in amniotic fluid that is increased in spontaneous preterm labor with intra-amniotic infection. Am. J. Reprod. Immunol. 80(4), e13031 (2018).
Para, R. et al. Human β-defensin-3 participates in intra-amniotic host defense in women with labor at term, spontaneous preterm labor and intact membranes, and preterm prelabor rupture of membranes. J. Matern. Fetal Neonatal. Med. 33(24), 4117–4132 (2020).
Galaz, J. et al. Cellular immune responses in amniotic fluid of women with preterm clinical chorioamnionitis. Inflamm. Res. 69(2), 203–216 (2020).
Gomez-Lopez, N. et al. Neutrophil extracellular traps in the amniotic cavity of women with intra-amniotic infection: A new mechanism of host defense. Reprod. Sci. 24(8), 1139–1153 (2017).
Gomez-Lopez, N., et al., Neutrophil extracellular traps in acute chorioamnionitis: A mechanism of host defense. Am. J. Reprod. Immunol. 77(3) (2017).
Gomez-Lopez, N. et al. The immunophenotype of amniotic fluid leukocytes in normal and complicated pregnancies. Am. J. Reprod. Immunol. 79(4), e12827 (2018).
Galaz, J. et al. Cellular immune responses in amniotic fluid of women with a sonographic short cervix. J. Perinat. Med. 48(7), 665–676 (2020).
Chan, R. L. Biochemical markers of spontaneous preterm birth in asymptomatic women. Biomed. Res. Int. 2014, 164081 (2014).
Funding
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. Maureen McGerty (Wayne State University) is also acknowledged for proofreading and editing the manuscript.
Author information
Authors and Affiliations
Contributions
A.L.T. and R.R. designed the study. D.W.G., B.D. and A.L.T. performed data analysis. D.W.G., A.L.T., N.G-L., J.G. and R.R. wrote the manuscript. G.B., B.D., E.J., S.M.B., D.G., M.B., M.S., R.D.P., C.P., F.G., T.C. provided feedback and suggestions for edits.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Gudicha, D.W., Romero, R., Gomez-Lopez, N. et al. The amniotic fluid proteome predicts imminent preterm delivery in asymptomatic women with a short cervix. Sci Rep 12, 11781 (2022). https://doi.org/10.1038/s41598-022-15392-3
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-022-15392-3
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
