A molecular signature of an arrest of descent in human parturition




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.



TABLE 1

Demographic and clinical characteristics of the study 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

Values are expressed as percentage (number) or median (interquartile range)

BMI , body mass index; qRT-PCR , quantitative real-time reverse transcription–polymerase chain reaction; NS , not significant.

Mittal. A myometrial transcriptome of an arrest of descent. Am J Obstet Gynecol 2011.

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.



TABLE 2

List of top 100 microarray probes with differential expression between myometrium from women with an arrest of descent compared with those with spontaneous term labor







































































































































































































































































































































































































































































































































































































































































































































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

Only gold members can continue reading. Log In or Register to continue

Jun 21, 2017 | Posted by in GYNECOLOGY | Comments Off on A molecular signature of an arrest of descent in human parturition
Premium Wordpress Themes by UFO Themes