Objective
This study was undertaken to identify the molecular basis of an arrest of descent.
Study Design
Human myometrium was obtained from women in term labor (TL; n = 29) and arrest of descent (AODes; n = 21). Gene expression was characterized using Illumina HumanHT-12 microarrays. A moderated Student t test and false discovery rate adjustment were applied for analysis. Confirmatory quantitative reverse transcription–polymerase chain reaction and immunoblot were performed in an independent sample set.
Results
Four hundred genes were differentially expressed between women with an AODes compared with those with TL. Gene Ontology analysis indicated enrichment of biological processes and molecular functions related to inflammation and muscle function. Impacted pathways included inflammation and the actin cytoskeleton. Overexpression of hypoxia inducible factor-1a, interleukin -6, and prostaglandin-endoperoxide synthase 2 in AODes was confirmed.
Conclusion
We have identified a stereotypic pattern of gene expression in the myometrium of women with an arrest of descent. This represents the first study examining the molecular basis of an arrest of descent using a genome-wide approach.
The common pathway of parturition is a complex process involving concomitant myometrial activation, cervical ripening, and membrane-decidual activation. Whereas the process of labor is vital to the survival of viviparous species, its physiology and pathology is incompletely understood. Dysfunctional term labor failure to dilate and/or descend) necessitates surgical intervention for delivery. Indeed, the frequent diagnosis of labor arrest disorders contributed to the performance of primary cesarean section rate of 23.5% of parturients in the United States in 2006.
Labor arrest disorders are often attributed to cephalopelvic disproportion. However, although cephalopelvic disproportion contributes to arrest of descent and dilatation, a subset of women who undergo cesarean section for an arrest disorder subsequently deliver a larger neonate vaginally. It is more likely that “failure to progress” represents a functional disorder of labor whose etiology is yet to be elucidated. Such functional disorders may result from inadequate or uncoordinated activation of the common pathway of parturition. Evidence suggests that in natural preparation for labor, the myometrium attains an increasingly contractile phenotype, whereas the cervix also undergoes preparatory changes. Insufficient preparation of the uterus or cervix for labor may prevent the successful coordinated efforts necessary for normal parturition.
High-dimensional biology techniques such as genomics, transcriptomics, and proteomics can be applied to determine the molecular signatures of both pathologic and physiologic states and provide insight into the biological processes involved. Although the transcriptome (tissue specific) of myometrium in normal term labor has been investigated, that of an arrest of descent has never been reported. We undertook this study to characterize the differential gene expression of human myometrium in patients with an arrest of descent and to explore the mechanisms leading to this common labor disorder.
Materials and Methods
A prospective study was performed in which human myometrium was obtained from women undergoing primary cesarean section at term (>37 weeks’ gestation) in the following groups: term spontaneous labor (n = 29); and arrest of descent (n = 21). Labor was diagnosed in the presence of spontaneous regular uterine contractions occurring at a minimum frequency of 2 every 10 minutes with cervical change that required hospital admission. Women in the term labor group underwent cesarean section because of a nonreassuring fetal status as determined by the primary physician or fetal malpresentation.
The diagnosis of arrest of descent was made in patients with complete cervical dilation without continued fetal descent after more than 1 hour. Only patients presenting in spontaneous labor were included. The placentas of all participating women were examined by an experienced pathologist (C.J.K.) who was blinded to the clinical diagnosis. Patients with clinical or histological chorioamnionitis and those undergoing labor induction were excluded.
All women provided written informed consent prior to the collection of myometrial samples. The collection and utilization of the samples for research purposes was approved by the Institutional Review Board of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (National Institutes of Health/Department of Health and Human Services, Bethesda, MD) and the Human Investigation Committees of Wayne State University (Detroit, MI) and the Sotero del Rio Hospital (Santiago, Chile).
Sample collection
Samples of myometrium were obtained from the lower uterine segment at the time of cesarean section, following delivery of the placenta. The biopsies were obtained from the midpoint of the superior aspect of the uterine incision using Metzenbaum scissors. All specimens measured approximately 1.0 × 1.0 × 1.0 cm. Tissue was ground under liquid nitrogen, placed in TRI Reagent (Applied Biosystems Inc, Foster City, CA) and kept at –80°C until analysis.
Total RNA extraction
Total ribonuclease acid (RNA) was isolated from snap-frozen myometrium using TRI Reagent combined with the QIAGEN RNeasy Lipid Tissue kit protocol (Qiagen, Valencia, CA) according to the manufacturer’s recommendation. The RNA concentrations and the A260/A280 nm ratio were assessed using a NanoDrop 1000 (Thermo Scientific, Wilmington, DE). RNA integrity numbers (RINs) were determined using the Bioanalyzer 2100 (Agilent Technologies, Wilmington, DE). An A260/A280 nm ratio of 1.66, a 28S/18S ratio of 0.2, and a RIN of 3.8 were minimum requirements for inclusion in expression analysis.
Microarray experiments
The Illumina HumanHT-12 version 3 expression microarray (Illumina, San Diego, CA) platform was used to measure the expression levels in each unpooled specimen per the manufacturer’s instructions. In brief, after purification of RNA using an RNeasy Mini Kit (Qiagen), 500 ng of total RNA was amplified and biotin labeled with the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX). Labeled complementary RNAs were hybridized to the Illumina HumanHT-12 version 3 expression BeadChip and imaged using a BeadArray reader. Raw data were obtained with BeadStudio software (Illumina).
Quantitative real-time reverse transcription–polymerase chain reaction (qRT-PCR)
A separate set of specimens for each group (term labor, n = 10; arrest of descent, n = 7) were obtained for qRT-PCR assays of select genes differentially expressed by microarray analysis. Total RNA (3 μg) was reverse transcribed using the SuperScript III First-Strand Synthesis System and oligo(dT) 20 primers (Invitrogen, Carlsbad, CA). PCR analyses were performed with TaqMan gene expression assays (hypoxia inducible factor-1a [HIF1-A]: Hs00936368_m1; interleukin [IL]-6: Hs00174131_m1; prostaglandin-endoperoxide synthase 2 [PTGS2]: Hs01573471_m1; adenosine triphosphatase, Na+/K+ transporting, alpha 1 polypeptide [ATP1A1]: Hs00167556_m1; G protein-coupled receptor 4 [GPR4]: Hs00947870_m1; calponin 1 [CNN1]: Hs00154543_m1; Caldesmon 1 [CALD1]: Hs00189021m1; Endo/exonuclease (5′-3′), endonuclease G-like [EXOG]: Hs00270782_m1, Filamin C, gamma [FLNC]: Hs00155124_m1; myosin light chain kinase [MYLK]: Hs00364926_m1, secretory leukocute peptidase inhibitor [SLPI]: Hs01070946_m1, super oxide dismutase 2 [SOD2]: Hs00167309_m1, and Sorbin and SH3 domain containing 1 [SORBS1]: Hs00908953_m1; Applied Biosystems). The human large ribosomal protein (RPLO) TaqMan endogenous control (part no. 4326314E) was used as the housekeeping gene for relative quantification. The gene-specific TaqMan assays and the RPLO housekeeping gene were run in triplicate (50 ng) for each case to allow for the assessment of technical variability.
Enzyme-linked immunosorbent assay
The myometrial protein concentration of IL-6 was determined with a specific enzyme-linked immunoassay (R&D Systems, Inc, Minneapolis, MN) according to the manufacturer’s instructions (term labor, n = 6; arrest of descent, n = 5).
Statistical analysis
Clinical data. Statistical analysis of clinical data was performed with Kruskal-Wallis and Mann-Whitney U test for post hoc analysis, χ 2 , and Fisher’s exact tests. The statistical package used was SPSS version 12 (SPSS Inc, Chicago, IL). A P value of less than .05 was considered significant.
Microarray analysis. The Illumina BeadStudio software suite was used to extract raw gene expression values from the array images. Data quality was assessed based on Illumina’s positive and negative control probes on each array as well as by inspection of the distributions of probe intensities. Data were normalized using the quantile normalization method. Probes that were called present (detection P < .1) in at least 5 samples were retained for further analysis. A moderated Student t test implemented in the limma library of Bioconductor was applied to test differential expression, and a false discovery rate adjustment of the P value was performed to correct for multiple testing. The probes were considered significantly different if the adjusted P value was less than .1, and the fold change difference between groups was at least 1.5.
Gene Ontology (GO) analysis was conducted using an overrepresentation approach described elsewhere and implemented in the GOstats software package that uses an established approach to deal with the correlation of P values between related GO terms. Pathway analysis was performed on the Kyoto Encyclopedia of Genes and Genomes pathway database using both the overrepresentation approach and the Signaling Pathway Impact Analysis (SPIA). The SPIA impact analysis is a systems biology approach that takes into account the gene-gene signaling interactions as well as the magnitude and direction of gene expression changes to determine significantly impacted pathways.
qRT-PCR assays and enzyme-linked immunosorbent assay (ELISA). Thirteen genes among those differentially expressed by microarray analysis were selected for confirmation with qRT-PCR based on their rank in the list of all differentially expressed genes as well as biological plausibility. Data analysis was performed using an equal variance 2-sample 1-tailed Student t test based on the hypothesis provided by the microarray data. The qRT-PCR results were considered significant with a P < .05 and if the direction of gene expression change between the groups was concordant with the microarray data. The IL-6 concentrations were compared using the Mann-Whitney U test.
Results
Demographic and clinical characteristics of the study groups are displayed in Table 1 . There were no significant differences in gestational age at delivery among the groups.
| Demographic | Term labor microarray (n = 29) | Arrest of descent microarray (n = 21) | Term labor qRT-PCR (n = 9) | Arrest of descent qRT-PCR (n = 7) | P value |
|---|---|---|---|---|---|
| Maternal age, y | 29 (23.5–34) | 23.5 (21.3–30.3) | 21 (19.5–27.5) | 22 (19–29) | .04 a |
| BMI, kg/m 2 | 28.7 (23.2–33.3) | 23.9 (21.9–27.2) | 27.9 (22.6–35.2) | 33.3 (24.7–39) | .03 b |
| Nulliparity, % | 48 (14/29) | 70 (14/20) | 56 (5/9) | 43 (3/7) | NS |
| Smoking, % | 7 (2/29) | 5 (1/20) | 11 (1/9) | 0 (0/7) | NS |
| African-American, % | 52 (14/29) | 35 (7/20) | 89 (8/9) | 86 (6/7) | NS |
| Gestational age at delivery, wk | 38.7 (38–40.4) | 39.4 (38.8–40.8) | 40.1 (38.8–40.6) | 40.1 (39.4–40.4) | NS |
| Birthweight, g | 3200 (2945–3572) | 3650 (3332–3966) | 3240 (2982–3632) | 3410 (2660–3830) | .02 c |
a Post hoc analysis revealed a significant difference between the term labor microarray and term labor qRT-PCR groups ( P = .01);
b Post hoc analysis was significant for comparisons between term labor microarray vs arrest of descent microarray ( P = .02) and arrest of descent microarray vs arrest of descent PCR groups ( P = .01);
c Post hoc analysis demonstrated differences between the arrest of descent microarray group and the term labor microarray ( P = .003) and qRT-PCR ( P = .04) groups.
Microarray analysis
Four hundred genes were differentially expressed between the myometrium of women at term in labor and those with an arrest of descent. Table 2 lists the top 100 genes differentially expressed between the 2 study groups ranked by P values. The differential expression results are depicted in Figure 1 . The volcano plot ( Figure 1 , A) shows the magnitude vs the significance of gene expression changes. Principal component analysis based visualization of the microarray data ( Figure 1 , B) was performed as previously described. This visualization of the samples in a 3-dimensional plot allows for inspection of the within-group transcriptome variability and, partly, the between-group differences.
| Probe ID | Entrez gene ID | Symbol | Gene name | Fold change | P value a |
|---|---|---|---|---|---|
| 6580634 | 2828 | GPR4 | G protein-coupled receptor 4 | 1.95 | .0001 |
| 4210095 | 476 | ATP1A1 | ATPase, Na+/K+ transporting, alpha 1 polypeptide | 1.55 | .0002 |
| 3370164 | 476 | ATP1A1 | ATPase, Na+/K+ transporting, alpha 1 polypeptide | 1.51 | .0006 |
| 2650730 | 6781 | STC1 | Stanniocalcin 1 | 2.75 | .0006 |
| 6350184 | 8771 | TNFRSF6B | Tumor necrosis factor receptor superfamily, member 6b, decoy | 3.48 | .0006 |
| 1230630 | 26207 | PITPNC1 | Phosphatidylinositol transfer protein, cytoplasmic 1 | 1.98 | .0006 |
| 6960072 | 3329 | HSPD1 | Heat shock 60kDa protein 1 (chaperonin) | 1.55 | .0006 |
| 3060273 | 4504 | MT3 | Metallothionein 3 | 2.44 | .0006 |
| 2510201 | 339768 | ESPNL | Espin-like | –1.59 | .0006 |
| 1580161 | 81831 | NETO2 | Neuropilin (NRP) and tolloid (TLL)-like 2 | 1.59 | .0007 |
| 6620528 | 4501 | MT1X | Metallothionein 1X | 2.92 | .0007 |
| 1050746 | 81502 | HM13 | Histocompatibility (minor) 13 | 1.52 | .0009 |
| 5810762 | 5239 | PGM5 | Phosphoglucomutase 5 | –2.74 | .0009 |
| 4570008 | 9941 | EXOG | Endo/exonuclease (5′-3′), endonuclease G-like | 2.94 | .001 |
| 2230678 | 32 | ACACB | Acetyl-coenzyme A carboxylase beta | –1.81 | .001 |
| 2190255 | 8771 | TNFRSF6B | Tumor necrosis factor receptor superfamily, member 6b, decoy | 2.46 | .001 |
| 770703 | 9459 | ARHGEF6 | Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6 | –1.71 | .0011 |
| 380494 | 5239 | PGM5 | Phosphoglucomutase 5 | –2.73 | .0011 |
| 1820504 | 4830 | NME1 | Nonmetastatic cells 1, protein (NM23A) expressed in | 1.57 | .0013 |
| 4610433 | 51129 | ANGPTL4 | Angiopoietin-like 4 | 2.81 | .0013 |
| 1820279 | 90139 | TSPAN18 | Tetraspanin 18 | –2.73 | .0013 |
| 1400446 | 123 | PLIN2 | Perilipin 2 | 1.83 | .0013 |
| 2810692 | 729359 | PLIN4 | Perilipin 4 | –2.09 | .0013 |
| 4480112 | 5239 | PGM5 | Phosphoglucomutase 5 | –2.62 | .0014 |
| 6840156 | 2762 | GMDS | GDP-mannose 4,6-dehydratase | 1.65 | .0014 |
| 4810026 | 10205 | MPZL2 | Myelin protein zero-like 2 | 1.51 | .0014 |
| 6760246 | 25802 | LMOD1 | Leiomodin 1 (smooth muscle) | –2.40 | .0014 |
| 7150292 | 388610 | TRNP1 | TMF1-regulated nuclear protein 1 | –1.63 | .0014 |
| 460204 | 123 | PLIN2 | Perilipin 2 | 1.80 | .0014 |
| 1510468 | 80273 | GRPEL1 | GrpE-like 1, mitochondrial (E. coli) | 1.50 | .0014 |
| 2650524 | 6164 | RPL34 | Ribosomal protein L34 | 1.58 | .0014 |
| 6370133 | 10483 | SEC23B | Sec23 homolog B (S. cerevisiae) | 1.53 | .0015 |
| 6620379 | 55222 | LRRC20 | Leucine rich repeat containing 20 | –1.61 | .0015 |
| 4180324 | 115572 | FAM46B | Family with sequence similarity 46, member B | –2.60 | .0015 |
| 3610193 | 64321 | SOX17 | SRY (sex determining region Y)-box 17 | 1.63 | .0015 |
| 1400634 | 4499 | MT1M | Metallothionein 1M | 2.73 | .0015 |
| 2640392 | 8771 | TNFRSF6B | Tumor necrosis factor receptor superfamily, member 6b, decoy | 1.87 | .0016 |
| 6280133 | 4629 | MYH11 | Myosin, heavy chain 11, smooth muscle | –1.59 | .0016 |
| 6860176 | 139411 | PTCHD1 | Patched domain containing 1 | –2.30 | .0016 |
| 2000292 | 92304 | SCGB3A1 | Secretoglobin, family 3A, member 1 | 1.58 | .0016 |
| 3940435 | 2012 | EMP1 | Epithelial membrane protein 1 | 1.51 | .0016 |
| 2480544 | 3779 | KCNMB1 | Potassium large conductance calcium-activated channel, Subfamily M, beta member 1 | –2.07 | .0016 |
| 60255 | 84168 | ANTXR1 | Anthrax toxin receptor 1 | –1.51 | .0017 |
| 70592 | 7414 | VCL | Vinculin | –1.59 | .0017 |
| 6420630 | 51435 | SCARA3 | Scavenger receptor class A, member 3 | –1.78 | .0017 |
| 5690167 | 6405 | SEMA3F | Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3F | 1.60 | .0018 |
| 1770333 | 8654 | PDE5A | Phosphodiesterase 5A, cGMP-specific | –1.62 | .0019 |
| 3610372 | 84319 | C3orf26 | Chromosome 3 open reading frame 26 | 1.93 | .002 |
| 650689 | 25999 | CLIP3 | CAP-GLY domain containing linker protein 3 | –1.86 | .002 |
| 1710692 | 23026 | MYO16 | Myosin XVI | –1.55 | .002 |
| 580403 | 1687 | DFNA5 | Deafness, autosomal dominant 5 | –1.83 | .002 |
| 1500241 | 10580 | SORBS1 | Sorbin and SH3 domain containing 1 | –2.46 | .002 |
| 6860424 | 6318 | SERPINB4 | Serpin peptidase inhibitor, clade B (ovalbumin), member 4 | 1.81 | .002 |
| 5260095 | 2706 | GJB2 | Gap junction protein, beta 2, 26 kDa | 1.90 | .002 |
| 1570047 | 6545 | SLC7A4 | Solute carrier family 7 (cationic amino acid transporter, y+ system), member 4 | –1.75 | .002 |
| 7200427 | 8862 | APLN | Apelin | 1.64 | .002 |
| 3140520 | 7111 | TMOD1 | Tropomodulin 1 | 1.66 | .002 |
| 6100482 | 493 | ATP2B4 | ATPase, Ca++ transporting, plasma membrane 4 | –1.94 | .002 |
| 1470056 | 6317 | SERPINB3 | Serpin peptidase inhibitor, clade B (ovalbumin), member 3 | 2.35 | .002 |
| 5890064 | 800 | CALD1 | Caldesmon 1 | –2.51 | .002 |
| 3140603 | 6840 | SVIL | Supervillin | –1.88 | .002 |
| 1170300 | 4495 | MT1G | Metallothionein 1G | 3.20 | .0021 |
| 6200402 | 4489 | MT1A | Metallothionein 1A | 2.36 | .0021 |
| 580709 | 1673 | DEFB4 | Defensin, beta 4 | 1.57 | .0021 |
| 450553 | 79026 | AHNAK | AHNAK nucleoprotein | –1.68 | .0021 |
| 5270519 | 115701 | ALPK2 | Alpha-kinase 2 | –2.24 | .0021 |
| 4570670 | 6237 | RRAS | Related RAS viral (r-ras) oncogene homolog | –1.77 | .0023 |
| 1340192 | 65055 | REEP1 | Receptor accessory protein 1 | –2.10 | .0023 |
| 580491 | 2318 | FLNC | Filamin C, gamma | –2.37 | .0023 |
| 1030239 | 9805 | SCRN1 | Secernin 1 | –1.59 | .0023 |
| 3190609 | 22904 | SBNO2 | Strawberry notch homolog 2 (Drosophila) | 1.55 | .0024 |
| 4280010 | 55679 | LIMS2 | LIM and senescent cell antigen-like domains 2 | –2.05 | .0024 |
| 4670441 | 10516 | FBLN5 | Fibulin 5 | –1.68 | .0025 |
| 2970279 | 7881 | KCNAB1 | Potassium voltage-gated channel, shaker-related subfamily, beta member 1 | –2.17 | .0025 |
| 50192 | 2532 | DARC | Duffy blood group, chemokine receptor | 1.90 | .0025 |
| 7330097 | 104 | ADARB1 | Adenosine deaminase, RNA-specific, B1 (RED1 homolog rat) | –1.56 | .0025 |
| 1300286 | 4638 | MYLK | Myosin light chain kinase | –2.26 | .0025 |
| 270292 | 2280 | FKBP1A | FK506 binding protein 1A, 12kDa | 1.70 | .0025 |
| 5910440 | 26872 | STEAP1 | Six transmembrane epithelial antigen of the prostate 1 | 2.16 | .0026 |
| 1940504 | 2280 | FKBP1A | FK506 binding protein 1A, 12kDa | 1.61 | .0026 |
| 5090315 | 1804 | DPP6 | Dipeptidyl-peptidase 6 | –1.86 | .0027 |
| 6220543 | 3091 | HIF1A | Hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor) | 1.53 | .0028 |
| 360053 | 65258 | MPPE1 | Metallophosphoesterase 1 | –1.57 | .0028 |
| 2640025 | 3240 | HP | Haptoglobin | 2.55 | .0029 |
| 7400286 | 3159 | HMGA1 | High mobility group AT-hook 1 | 1.76 | .003 |
| 5690639 | 10516 | FBLN5 | Fibulin 5 | –1.62 | .003 |
| 6560564 | 5578 | PRKCA | Protein kinase C, alpha | –1.53 | .0031 |
| 5690139 | 80310 | PDGFD | Platelet derived growth factor D | –1.54 | .0031 |
| 6580056 | 27189 | IL17C | Interleukin 17C | 1.99 | .0032 |
| 5960682 | 348093 | RBPMS2 | RNA binding protein with multiple splicing 2 | –1.96 | .0032 |
| 1710735 | 23002 | DAAM1 | Dishevelled associated activator of morphogenesis 1 | –1.85 | .0032 |
| 6380669 | 6695 | SPOCK1 | Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1 | –1.51 | .0032 |
| 3390551 | 113146 | AHNAK2 | AHNAK nucleoprotein 2 | –2.42 | .0032 |
| 2340241 | 3613 | IMPA2 | Inositol(myo)-1(or 4)-monophosphatase 2 | –1.77 | .0033 |
| 5080364 | 87 | ACTN1 | Actinin, alpha 1 | –1.53 | .0033 |
| 6420050 | 4885 | NPTX2 | Neuronal pentraxin II | 2.11 | .0033 |
| 10543 | 139728 | PNCK | Pregnancy up-regulated nonubiquitously expressed CaM kinase | –2.50 | .0034 |
| 670386 | 3397 | ID1 | Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein | 1.67 | .0034 |
| 2230379 | 10135 | NAMPT | Nicotinamide phosphoribosyltransferase | 2.44 | .0034 |
| 10296 | 1674 | DES | Desmin | –2.98 | .0034 |
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