Characterization of the transcriptome of chorioamniotic membranes at the site of rupture in spontaneous labor at term

Abstract

OBJECTIVE

The purpose of this study was to compare the transcriptome between the site of membrane rupture and the chorioamniotic membranes away from the site of rupture.

STUDY DESIGN

The transcriptome of amnion and chorion (n =20 each) from and distal to the site of rupture from women with spontaneous labor and vaginal delivery at term after spontaneous rupture of membranes was profiled with Illumina HumanHT-12 microarrays. Selected genes were validated with the use of quantitative reverse transcription–polymerase chain reaction.

RESULTS

Six hundred seventy-seven genes were differentially expressed in the chorion between the rupture and nonrupture sites (false discovery rate <0.1; fold change >1.5). Quantitative reverse transcription–polymerase chain reaction confirmed the differential expression in 10 of 14 genes. Enriched biological processes included anatomic structure development, cell adhesion and signal transduction. Extracellular matrix–receptor interaction was the most impacted signaling pathway.

CONCLUSION

The transcriptome of fetal membranes after spontaneous rupture of membranes in term labor is characterized by region-and tissue-specific differential expression of genes that are involved in signature pathways, which include extracellular matrix–receptor interactions.

The common pathway of human parturition is characterized by anatomic, immunologic, and physiologic changes that occur in the mother and/or fetus in both term and preterm parturition.¹ In normal pregnancy, the chorioamniotic membranes have important anatomic,² biochemical,^3–5 and antibacterial^6,7 properties that serve to protect the fetus and pregnancy. The membranes (chorion and amnion) are intact during gestation and rupture without intervention close to the beginning of the second stage of labor.⁸

The fetal membranes undergo complex physical and biochemical changes before the onset of labor at term.^8–11 In patients at term without labor, the portion of the fetal membranes overlying the cervix contains characteristic histologic and biochemical changes and is thought to be the site at which eventual spontaneous rupture of membranes occurs.^2,5,10,12,13 This area has been termed the “zone of altered morphology” (ZAM).^2,10 Defined by histologic criteria, the membranes in the ZAM are thinner than in the membranes away from the ZAM. The features of the ZAM include an increase in the thickness of connective tissue of amnion and chorion and decreased thickness of the cytotro-phoblast and decidual layers.^10,12 Moreover, El Khwad et al¹³ demonstrated that the ZAM is a weak zone by using biophysical testing. Several mechanisms have been proposed to contribute to the weakening of the chorioamniotic membranes in the ZAM; these mechanisms include spontaneous separation of fetal membrane layers,¹⁴ the exposure of the membranes to cytokines and matrix-degrading enzymes that are present in the vagina^15–17 and in amniotic fluid^18–25 that are known to increase in normal pregnancy at term and in labor.

The use of high-dimensional biology technology has led to the identification of novel genes, proteins, and pathways that are involved in both term and pre-term labor.^26–28 Specifically, transcriptomics (the description of the entire set of genes that are transcribed by a particular tissue at a specific point in time) has been used to examine the differences in global gene expression profiles in intact fetal membranes of women at term with and without labor,²⁹ women in term and preterm labor,³⁰ and in pregnancies that are complicated by preterm labor with and without preterm prelabor rupture of membranes (PROM).³¹ Gene expression profiling of the chorioamniotic membranes in the context of spontaneous rupture of membranes (SROM) at the site of membrane rupture and away from the site of rupture has not been previously reported.

This study was undertaken to examine the differences in the transcriptome between the site of membrane rupture and a section of the intact chorioamniotic membranes away from the site of rupture in patients at term with spontaneous labor and to gain further insight into the biochemical changes that are associated with the phenomenon of membrane rupture.

Methods

Study group

A prospective study was designed to examine differential gene expression of the chorioamniotic membranes at the gross site of rupture and distant from the site of rupture; samples were obtained from women who were admitted with spontaneous labor at term with SROM (n = 30). The inclusion criteria were (1) gestational age of ≥37 weeks, (2) absence of medical or obstetric complications of pregnancy, (3) no antibiotic administration in labor for group beta streptococcus (GBS) prophylaxis, (4) absence of meconium staining of the amniotic fluid, (5) no prostaglandin administration, (6) no use of amnioinfusion, (7) vaginal birth, and (8) normal pregnancy outcome, including an infant who was of appropriate weight for gestational age without congenital anomalies and Apgar scores >7 at 1 and 5 minutes. All patients provided written informed consent for the collection and use of samples for research purposes under the protocols approved by the institutional review boards of Wayne State University and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services.

Clinical definitions

Labor was defined as the presence of regular uterine contractions accompanied by progressive cervical dilation that led to delivery. SROM was defined as rupture of membranes in the presence of labor and confirmed with sterile speculum examination and a combination of pooling, ferning, and Nitrazine tests. An appropriate-for-gestational-age neonate was defined as a birthweight between the 10th and 90th percentile for the gestational age at birth.³² Histologic chorio-amnionitis was diagnosed on the basis of the presence of inflammatory cells in the chorionic plate and/or chorioamniotic membranes.^33,34 Acute funisitis was diagnosed by the presence of neutrophils in the wall of the umbilical vessels and/or Wharton’s jelly.

Collection of the fetal membranes

Fetal membranes were processed within 30 minutes of delivery. After the identification of the site of rupture, a linear piece of membrane that measured 1 ×4 cm and spanned the gross rupture site was dissected sharply (Figure 1). Similarly, a 1 ×4 cm strip of reflected extraplacental membranes was removed at a site distal to the site of rupture that was approximately one-half the distance between the site of rupture and the placental disk.^35,36 The amnion was separated from the chorion, and each tissue was flash-frozen with liquid nitrogen. This method yielded a set of 4 specimens from each patient: (1) amnion from the rupture site, (2) chorion from the rupture site, (3) amnion from the nonrupture site, and (4) chorion from the non-rupture site. In addition, a section of membranes from each patient was obtained for histologic examination with the use of hematoxylineosin–stained histologic sections. The cases with chorioamnionitis, funisitis, meconium-staining, or severe amnion degeneration were excluded from the study.^33,34 The sets of specimens were collected prospectively until 30 total sets of fetal membranes were obtained that met all inclusion and histologic criteria. The RNA was extracted and stored at −70°C until it was assayed. The materials were housed at the bank of biologic specimens that was held at the Perinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development.

FIGURE 1. Sampling sites of the fetal membranes.

The image of the placenta demonstrates the representative sampling sites of the gross site of rupture (yellow box) and the site away from rupture (white box).

Microarray analysis and real-time quantitative polymerase chain reaction

For microarray analysis, complete sets of chorioamniotic membranes were used (n =20 matched sets of amnion from the rupture site, chorion from the rupture site, amnion from the nonrupture site, and chorion from the nonrupture site from each patient). The Illumina HumanHT-12 expression microarray (version 3; Illumina, San Diego, CA) platform was used to measure the gene expression levels in each specimen, per manufacturer’s instructions. To verify the results from microarray, 14 genes were selected for quantitative polymerase chain reaction (qRT-PCR) assays in a subset of the original sample set (n =10) and in a new set of samples from patients at term with SROM (n =10). The selection criteria for the new specimens were the same as those used to select patients for the microarray analysis, with the exception of 2 patients with mild intermittent asthma without asthma exacerbations or exposure to steroids during the index pregnancy. A detailed description of the method and analysis is available as supplementary material (Supplement 1).

Statistical analyses

The raw gene expression data were normalized by a quantile normalization approach.³⁷ A paired moderated t test was used to test for differential expression with a false discovery rate (FDR)³⁸ threshold of 0.1 in conjunction with a threshold of 1.5 on the fold change to assign gene significance.³⁹ Gene ontology analysis was performed with algorithms that were described previously.⁴⁰ Pathway analysis was performed on the Kyoto Encyclopedia of Genes and Genomes (KEGG)⁴¹ pathway database with an overrepresentation analysis⁴² and the signaling pathway impact analysis.^43,44 The signaling pathway impact analysis differs from the overrepresentation approaches by taking into account the gene-gene signaling interactions and the magnitude and direction of gene expression changes to determine significantly impacted pathways.

Differential expression between experimental regions from qRT-PCR data was performed with a paired t test on −ΔCt values. The Student t, Mann-Whitney U, and χ² tests were used to identify significant differences in patient demographics between women in the microarray and qRT-PCR groups. SPSS software (version 12.0; SPSS Inc, Chi-cago, IL) was used for statistical analysis of demographic data. A probability value of<.05 was considered statistically significant.

Results

Demographics

Table 1 displays the demographic characteristics of patients who were included in the microarray and qRT-PCR analyses.

TABLE 1.

Patient demographics

Demographic	Microarray (n =20)	Quantitative reverse transcriptase–polymerase chain reaction (n = 10)	P value
Maternal age, ya	24.5 (20.3–27.8)	26 (20–30)	NSb
Gravidity, na	3 (2–4)	2.5 (2–4.5)	NSc
Parity, na	1 (0–2)	1 (0–2.8)	NSc
Maternal race, %			NSd
African American	80	70
White	10	10
Other	10	20
Gestational age at delivery, wka	38.6 (38.2–40.1)	39 (38.2–40.3)	NSb
Rupture of membranes-to-delivery interval, mina	259 (104–624)	121 (59–189)	NSc
Birthweight, ga	3285 (3097–3360)	3335 (3203–3409)	NSb

NS, not significant.

^aValues expressed as medians (interquartile range);

^bStudent t test;

^cMann-Whitney U test;

^dχ² test.

Results of the microarray analysis

Rupture site chorion vs nonrupture site chorion

Microarray analysis demonstrated 815 probes that corresponded with 677 unique genes that were expressed differentially in the chorion between the rupture and nonrupture sites (pFDR <0.1; fold change >1.5). A total of 453 genes had decreased expression, and 224 genes had increased expression in the rupture site, compared with the nonrupture site. A “volcano plot” shows the differential expression of all the annotated probes on the Illumina HumanHT-12 v3 array with the log (base 10) of the FDR-adjusted probability values (y-axis) plotted against the log (base 2) fold changes (x-axis) between the rupture site chorion and nonrupture site chorion (Figure 2). A heat map that displays the hierarchical clustering of all samples and 200 probes whose expression had the largest variance across all amnion and chorion samples (both rupture and nonrupture sites) is shown in Figure 3, A. Two main clusters are seen in the heat map that predominantly represent amnion from the rupture and non-rupture site in 1 cluster and chorion from the rupture and nonrupture site in the other cluster. A smaller cluster of mixed samples (both amnion and chorion and rupture vs nonrupture site specimens) is also seen in the heat map and represents specimens that did not fit into the 2 main clusters of specimens. Figure 3, B, shows a hierarchical clustering of the 200 probes with the highest variance across only chorion specimens (both rupture and nonrupture site). A list of the top 100 differentially expressed genes between the rupture site chorion and nonrupture site chorion is presented in Table 2; the complete list of differentially expressed probes is available as supplementary material (Supplement 2).

FIGURE 2. Transcriptomic analysis of chorion specimens.

The volcano plot shows probability values of all probes in the microarray plotted against the fold changes. In this Figure, the log (base 10) of the false discovery rate–adjusted probability values are plotted against the log (base 2) ratio of fold changes between ruptured site chorion and nonruptured site chorion. On the Y-axis, values higher than the grey-line threshold represent significant probes with an adjusted probability value of < .1. On the X-axis, values outside the red lines represent fold-changes of ≥1.5 between ruptured site chorion and nonruptured site chorion. The 815 differentially expressed probes are labeled blue in the upper outer quadrants.

FIGURE 3. Heat maps of the clustering of genes.

A, A heat map displays the hierarchical clustering of the 200 probes whose expression had the largest variance across all amnion and chorion specimens. The red and green colors in the heat map correspond with high and low gene expression, respectively. The labels at the top indicate individual patient samples and their membrane region designation. Two main clusters are seen that predominantly represent amnion from the rupture site and nonrupture site on the left and chorion from the rupture site and nonrupture site in the middle. A smaller cluster of mixed samples are on the right. B, A heat map displays the hierarchical clustering of the 200 probes whose expression had the largest variance across chorion specimens. Two separate cluster regions represent the rupture site on the right and non-rupture site on the left.

TABLE 2.

List of the top 100 differentially expressed probes in chorion between the rupture site and nonruptured site

Rank	Name	Entreza	Symbolb	Fold changec	P value
1	Methyltransferase like 7A	25840	METTL7A	−1.9	.000023
2	Epoxide hydrolase 1, microsomal (xenobiotic)	2052	EPHX1	−1.7	.000048
3	Arylacetamide deacetylase-like 2	344752	AADACL2	−4.5	.000057
4	KIT ligand	4254	KITLG	−1.6	.000057
5	Glutathione S-transferase alpha 4	2941	GSTA4	−1.6	.000057
6	Killer cell lectin-like receptor subfamily C, member 1	3821	KLRC1	−1.7	.000069
7	Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)	6387	CXCL12	−1.7	.000100
8	Basic helix-loop-helix family, member e40	8553	BHLHE40	1.6	.000100
9	Aldehyde oxidase 1	316	AOX1	−3.3	.000100
10	Meis homeobox 1	4211	MEIS1	−1.8	.000100
11	Complement factor I	3426	CFI	−1.7	.000100
12	Tetraspanin 7	7102	TSPAN7	−2.2	.000110
13	Phosphoglucomutase 5	5239	PGM5	−3.3	.000120
14	G protein-coupled receptor, family C, group 5, member A	9052	GPRC5A	1.8	.000140
15	Arylacetamide deacetylase (esterase)	13	AADAC	−5.5	.000140
16	EGF-containing fibulin-like extracellular matrix protein 1	2202	EFEMP1	−2.2	.000140
17	Chromosome 12 open reading frame 48	55010	C12orf48	−1.8	.000140
18	B-cell CLL/lymphoma 2	596	BCL2	−2.3	.000140
19	Regulator of G-protein signaling 5	8490	RGS5	−2.0	.000140
20	BTB (POZ) domain containing 3	22903	BTBD3	−2.2	.000140
21	Phosphoribosyl pyrophosphate synthetase 2	5634	PRPS2	−1.7	.000140
22	Phosphoglucomutase 5	5239	PGM5	−3.3	.000140
23	Primary ciliary dyskinesia protein 1	200373	hCG_17324	−1.6	.000150
24	Killer cell lectin-like receptor subfamily C, member 1	3821	KLRC1	−2.1	.000150
25	Gtpase, IMAP family member 6	474344	GIMAP6	−2.2	.000150
26	Transmembrane 4 L 6 family member 18	116441	TM4SF18	−1.8	.000150
27	Interleukin 15	3600	IL15	−1.8	.000160
28	Lectin, galactoside-binding, soluble, 9	3965	LGALS9	−1.8	.000170
29	Plasticity related gene 1	9890	LPPR4	−1.6	.000180
30	Endo/exonuclease (5′-3′), endonuclease G-like	9941	EXOG	2.0	.000180
31	CD34 molecule	947	CD34	−3.3	.000180
32	MAP6 domain containing 1	79929	MAP6D1	1.6	.000180
33	Calcitonin receptor-like	10203	CALCRL	−1.9	.000180
34	Cathepsin W	1521	CTSW	−2.2	.000180
35	Toll-like receptor 7	51284	TLR7	−1.5	.000190
36	G protein-coupled receptor 34	2857	GPR34	−1.8	.000190
37	Keratin 14	3861	KRT14	3.0	.000190
38	Acetyl-coenzyme A acetyltransferase 1	38	ACAT1	−1.6	.000190
39	C-type lectin domain family 14, member A	161198	CLEC14A	−2.2	.000190
40	Similar to Complement C3 precursor	653879	LOC653879	−2.6	.000190
41	Phosphoglucomutase 5	5239	PGM5	−2.2	.000190
42	Chemokine (C-C motif) ligand 18 (pulmonary and activation- regulated)	6362	CCL18	−2.2	.000190
43	LIM domain binding 2	9079	LDB2	−2.1	.000190
44	GRB2-related adaptor protein	10750	GRAP	−1.9	.000190
45	B-cell CLL/lymphoma 2	596	BCL2	−1.9	.000190
46	Olfactomedin-like 1	283298	OLFML1	−1.6	.000190
47	CHK2 checkpoint homolog (S. Pombe)	11200	CHEK2	−1.6	.000190
48	Gtpase, IMAP family member 7	168537	GIMAP7	−2.4	.000190
49	Chromosome 5 open reading frame 4	10826	C5orf4	−1.9	.000190
50	Sterile alpha motif domain containing 3	154075	SAMD3	−1.6	.000190
51	Src kinase associated phosphoprotein 1	8631	SKAP1	−1.7	.000190
52	Tetraspanin 7	7102	TSPAN7	−2.5	.000190
53	Teashirt zinc finger homeobox 2	128553	TSHZ2	−2.1	.000190
54	Transcobalamin II; macrocytic anemia	6948	TCN2	−1.9	.000190
55	Purinergic receptor P2Y, G-protein coupled, 14	9934	P2RY14	−2.7	.000190
56	Family with sequence similarity 150, member B	285016	FAM150B	−3.1	.000200
57	Carboxypeptidase B1 (tissue)	1360	CPB1	−2.8	.000200
58	Regulator of G-protein signaling 22	26166	RGS22	−2.8	.000200
59	Uracil-DNA glycosylase	7374	UNG	−1.6	.000210
60	Phospholipid scramblase 4	57088	PLSCR4	−1.7	.000210
61	Dynein, cytoplasmic 2, light intermediate chain 1	51626	DYNC2LI1	−1.6	.000220
62	Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)	6387	CXCL12	−1.9	.000220
63	Uridine phosphorylase 1	7378	UPP1	1.6	.000220
64	Pyruvate dehydrogenase kinase, isozyme 4	5166	PDK4	−2.2	.000230
65	Nuclear factor I/B	4781	NFIB	−2.0	.000230
66	Regulator of G-protein signaling 5	8490	RGS5	−1.9	.000250
67	Chromosome 21 open reading frame 34	388815	C21orf34	−1.8	.000250
68	Pannexin 1	24145	PANX1	1.6	.000250
69	Acetyl-coenzyme A carboxylase beta	32	ACACB	−1.6	.000270
70	CHK2 checkpoint homolog (S. Pombe)	11200	CHEK2	−1.6	.000280
71	Family with sequence similarity 5, member B	57795	FAM5B	−2.0	.000280
72	Integral membrane protein 2A	9452	ITM2A	−2.3	.000280
73	Chemokine (C-C motif) ligand 15	6359	CCL15	−2.2	.000290
74	ATP-binding cassette, sub-family A (ABC1), member 8	10351	ABCA8	−2.2	.000290
75	Lectin, galactoside-binding, soluble, 9	3965	LGALS9	−2.0	.000290
76	Phosphoinositide-3-kinase, regulatory subunit 1 (alpha)	5295	PIK3R1	−1.7	.000290
77	Gap junction protein, alpha 4, 37 kd	2701	GJA4	−1.6	.000290
78	Sorting nexin family member 21	90203	SNX21	−1.6	.000300
79	Phosphoribosyl pyrophosphate synthetase 2	5634	PRPS2	−1.5	.000300
80	LRRN4 C-terminal like	221091	LRRN4CL	−1.8	.000310
81	Transmembrane protein 27	57393	TMEM27	−2.0	.000340
82	CD34 molecule	947	CD34	−3.2	.000340
83	Von Willebrand factor	7450	VWF	−4.3	.000350
84	Major histocompatibility complex, class II, DO alpha	3111	HLA-DOA	−1.9	.000360
85	Granzyme A (granzyme 1)	3001	GZMA	−2.9	.000380
86	Transforming growth factor, beta-induced, 68 kd	7045	TGFBI	1.6	.000390
87	Nuclear factor I/B	4781	NFIB	−2.0	.000390
88	Galactosidase, beta 1-like 2	89944	GLB1L2	−2.3	.000390
89	Endoplasmic reticulum metallopeptidase 1	79956	ERMP1	−1.5	.000400
90	Serum deprivation response (phosphatidylserine binding protein)	8436	SDPR	−2.2	.000410
91	ABI family, member 3	51225	ABI3	−1.6	.000420
92	Sterol-C4-methyl oxidase-like	6307	SC4MOL	1.6	.000430
93	Solute carrier family 7 (cationic amino acid transporter, y3 system), member 5	8140	SLC7A5	1.9	.000430
94	Src homology 2 domain containing E	126669	SHE	−1.5	.000440
95	Similar to Complement C3 precursor	653879	LOC653879	−2.2	.000440
96	Ras homolog gene family, member J	57381	RHOJ	−1.9	.000440
97	Membrane-spanning 4-domains, subfamily A, member 4	51338	MS4A4A	−1.7	.000440
98	Hypothetic protein LOC100133666	100133666	LOC100133666	−1.6	.000440
99	Frizzled homolog 8 (Drosophila)	8325	FZD8	1.7	.000440
100	Osteoglycin	4969	OGN	−2.7	.000440

ABI, Abelson-interactor; ATP, adenosine triphospate; BTB, Broad-complex, Tramtrack and Bric-abrac; CD34, cluster of differentiation 34; CHK2, checkpoint kinase 2; CLL, chronic lymphocytic leukemia; EGF, epidermal growth factor; GRB2, growth factor receptor-bound protein 2; IMAP, immunity-associated protein; KIT, c-Kit; LRRN4, leucine rich repeat neuronal 4; MAP6, micortubule-associated protein 6; POZ, poxvirus and zinc finger.

^aEntrez gene identification;

^bSymbol was taken from the gene database and corresponds to official Human Genome Organization Gene Nomenclature Committee symbols;

^cfold change (the number of times the average expression level in the ruptured sites is different from the average expression level in the nonruptured sites) refers to probe expression change in ruptured chorion relative to nonruptured chorion; positive values (no sign) indicate increased expression, and negative values (minus sign) indicate decreased expression.

Gene ontology metaanalysis of the significantly up- and down-regulated genes was performed to identify gene ontology terms that were represented by the differentially expressed genes. In this analysis, 130 biologic processes were enriched (pFDR, <0.05); the top 20 processes are presented in Table 3.

TABLE 3.

Top 20 biological processes with enrichment in chorion between the rupture site and the nonrupture site

Rank	Biological process	Genes in differentially expressed list, n	Genes in reference array, n	Adjusted P value
1	Anatomic structure development	151	1785	< .0001
2	Multicellular organismal process	202	2681	< .0001
3	Immune response	61	520	< .0001
4	Multicellular organismal development	152	1919	< .0001
5	Response to external stimulus	66	596	< .0001
6	Epidermis development	25	118	< .0001
7	Antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex class II	10	16	< .0001
8	Cell adhesion	65	591	< .0001
9	Biologic adhesion	65	591	< .0001
10	Developmental process	196	2720	< .0001
11	Organ development	98	1074	< .0001
12	Cell communication	211	3006	< .0001
13	Ectoderm development	26	132	< .0001
14	Signal transduction	194	2739	< .0001
15	Anatomic structure morphogenesis	89	971	< .0001
16	System development	122	1496	< .0001
17	Response to wounding	47	381	< .0001
18	Response to stimulus	157	2100	< .0001
19	Immune system process	72	753	< .0001
20	Locomotion	42	346	< .0001

Pathway analysis of the significant genes was undertaken with an overrepresentation method and a signaling pathway impact analysis method. Using the overrepresentation method, 14 KEGG pathways were significant (pFDR, <0.05) in the comparison between rupture site chorion and nonrupture site chorion (Table 4). The 5 most significant pathways were (1) cell junctions, (2) graft-vs-host disease, (3) complement and coagulation cascades, (4) extracellular matrix–receptor interaction, and (5) allograft rejection. Impact analysis of the KEGG signaling pathways also identified 14 significant pathways (Figure 4), although the ranking of the most impacted pathways were different than the ranking of the pathways from the overrepresentation method. The most impacted pathway was the extracellular matrix–receptor interaction. Moreover, the signaling pathway impact method identified 4 pathways that were not identified by the overrepresentation method: cytokine-cytokine receptor interaction, gap junction, pathogenic Escherichia coli infection, and adipocytokine signaling.

TABLE 4.

Significant pathways with overrepresentation analysis in chorion between rupture site and nonrupture site

Rank	Map name	Kyoto Encyclopedia of Genes and Genomes identification number	Genes in differentially expressed list, n	Genes in reference array, n	Adjusted P value
1	Cell junctions	1430	30	108	< .0001
2	Graft-vs-host disease	5332	17	38	< .0001
3	Complement and coagulation cascades	4610	17	53	< .0001
4	Extracellular matrix–receptor interaction	4512	20	73	< .0001
5	Allograft rejection	5330	13	32	< .0001
6	Cell adhesion molecules	4514	25	115	< .0001
7	Type I diabetes mellitus	4940	13	38	< .0001
8	Asthma	5310	10	23	< .0001
9	Focal adhesion	4510	30	184	< .0001
10	Autoimmune thyroid disease	5320	12	40	.0001
11	Antigen processing and presentation	4612	16	74	.0002
12	Hematopoietic cell lineage	4640	15	70	.0003
13	Systemic lupus erythematosus	5322	16	101	.0064
14	Metabolism of xenobiotics by cytochrome P450	980	9	43	.0130

FIGURE 4. Significantly impacted pathways in chorion specimens.

Two-dimensional plot illustrates the relationship between the 2 types of evidence considered by signaling pathway impact analysis. The X-axis shows the overrepresentation evidence (−log[P value]); the Y-axis shows the perturbation evidence (−log[P value]). Each pathway is represented by a point. Pathways above the oblique red line (red dots) are significant at 5% after Bonferroni correction; the pathways (blue dots and red dots) above the oblique blue line are significant at 5% after false discovery rate correction. The vertical and horizontal thresholds represent the same corrections for the 2 types of evidence considered individually.

Rupture site chorion vs rupture site amnion

In the comparison between rupture site chorion and rupture site amnion, transcriptional profiling demonstrated 2279 differentially expressed probes that corresponded with 1981 unique genes (pFDR <0.1; fold change >1.5). Gene ontology metaanalysis of the significant genes revealed the enrichment of 172 biological processes that include developmental process, anatomic structure development, cell adhesion, biologic adhesion, and multicellular organismal development. The over-representation analysis of genes identified 6 significant KEGG pathways: (1) cell junctions, (2) cell adhesion molecules, (3) complement and coagulation cascades, (4) extracellular matrix–receptor interaction, (5) focal adhesion, and (6) asthma. In contrast, signaling pathway impact analysis identified 12 significantly impacted pathways. The data are available as supplementary material (Supplement 3).

Nonrupture site chorion vs nonrupture site amnion

In the comparison between chorion and amnion from the nonruptured site, 3427 probes that represented 2849 genes were differentially expressed (pFDR <0.1; fold change >1.5, data not shown). There were 217 biological processes that were enriched; the top 5 biological processes were anatomic structure development, developmental process, cell adhesion, biologic adhesion, and organ development. Twelve overrepresented KEGG pathways and 17 significantly impacted pathways were identified in this comparison and are available as supplementary material (Supplement 3).

Rupture site amnion vs nonrupture site amnion

There were no significant regional differences in the amnion transcriptome (rupture vs nonrupture).

Augmentation of spontaneous labor

In spontaneous labor at term, there was no difference in the transcriptome of the chorion of patients who underwent augmentation with oxytocin, when compared with patients without augmentation. In contrast, 4 genes were expressed differentially when amnion was compared with and without exposure to oxytocin augmentation: (1) AGA (1.5-fold decrease; pFDR <0.01); (2) RRAGD (1.8-fold increase; pFDR <0.01); (3) LGALS7B (4.2-fold increase; pFDR <0.01); and (4) SORD2 (1.8-fold increase; pFDR <0.01). No significant biological processes or pathways were identified between the amnion groups. These data were not validated with qRT-PCR.

qRT-PCR analysis

Rupture site chorion vs nonrupture site chorion

The results of qRT-PCR of a random subset of the original specimens (n =10) confirmed the differential expression of 11 of 14 genes that were found to be significant on the microarray analysis (Table 5). In a new set of specimens (n =10), qRT-PCR validated the microarray results in 10 of 14 genes (Figure 5). Specifically, qRT-PCR confirmed increased expression of IL6 and PTGS2. Consistent with the microarray analysis, qRT-PCR revealed decreased expression of PAEP, GNLY, CXCL12, CXCL14, IGFBP2, IGFBP4, CNR1, and ADAMTS5 in the rupture site, compared with the nonrupture site. Combining the 2 sets of patients (n =20) for analysis, qRT-PCR validated both the direction of fold change and the significance of 13 of 14 of the differentially expressed genes selected from the microarray analysis.

TABLE 5.

Comparison of microarray data to quantitative reverse transcriptase–polymerase chain reaction results of selected genes in original study samples and in a new replication set in chorion between rupture site and nonrupture site

Gene symbol	Original sample set: array fold change (n = 20)	Quantitative reverse transcriptase–polymerase chain reaction
		Original sample set (n = 10)a		New replication set (n = 10)		Combined sample set (n = 20)
		Fold change	P value	Fold change	P value	Fold change	P value
ADAMTS5	−3.17	−2.81	.0239	−2.20	.0816	−2.46	.0084
BCL2	−2.26	−5.47	.0190	−2.48	NS	−3.54	.0116
CNR1	−2.26	−2.64	.0681	−2.77	.0824	−2.71	.0188
IL1RN	3.05	2.37	.0965	3.71	NS	3.03	.0480
CXCL12	−1.72	−15.60	.0009	−10.58	.0139	−12.72	.0001
IL-6	2.71	2.66	NS	13.25	.0233	6.44	.0105
CXCL14	−2.32	−6.14	.0205	−2.83	.0545	−4.01	.0037
PTGS2	2.10	1.61	NS	3.45	.0213	2.45	.0090
GNLY	−4.40	−6.27	.0050	−6.31	.0123	−6.29	.0003
PAEP	−3.93	−24.15	.0072	−30.97	.0218	−27.53	.0008
IGF2	1.57	NA	NA	−1.00	NS	−1.00	NS
IGFBP2	−2.97	−3.51	.0156	−3.29	.0323	−3.39	.0020
IGFBP4	−1.50	−2.71	.0420	−3.07	.0166	−2.90	.0022
IGFBP6	−2.30	−3.13	.0394	−1.09	NS	−1.74	.0711

NA, not available; NS, not significant.

^aOf the 20 original specimens, 10 specimens were selected randomly for quantitative reverse transcriptase–polymerase chain reaction.

FIGURE 5. Quantitative reverse transcriptase–polymerase chain reaction results.

Results of quantitative reverse transcriptase–polymerase chain reaction assay of selected genes in a new set of specimens. The shaded boxes represent genes that are statistically significant (P< .05); the white boxes represent nonsignificant genes. The data are presented as the difference between the −ΔCt of rupture site chorion and the −ΔCt of non-rupture site chorion: positive values correlate with increased expression and negative values correlate with decreased expression of the gene in the rupture site chorion compared with the nonrupture site chorion. The boxes encompass 50% of the data from the 1st quartile to the 3rd quartile. The middle line represents the median value (50%) quartile. The whiskers extend to the most extreme data point but do not exceed values of >1.5 times the inter-quartile range from the box. The circles represent outliers.

Nonrupture site chorion vs nonrupture site amnion

In the comparison between nonrupture site chorion and nonrupture site amnion, gene expression was found to correspond with the microarray data for PTGS2 (9.4-fold decrease; P=.0044), IL1RN (69.9-fold decrease; P=.0039), and CXCL14 (76.4-fold increase; P=.002).

Rupture site chorion vs rupture site amnion

In the comparison between rupture site chorion and rupture site amnion, significant fold changes in messenger RNA (mRNA) expression were detected for PTGS2 (2.9-fold decrease; P=.011), IL1RN (30.2-fold decrease; P=.002), and CXCL14 (88-fold increase; P=.002).

Rupture site amnion vs nonrupture site amnion

Corresponding with the microarray analysis, PTGS2, IL1RN, and CXCL14 PCR results were not statistically significant in the comparisons between rupture site amnion and nonrupture site amnion.

Comment

Microarray analysis revealed 677 differentially expressed genes in the chorion of the fetal membranes between the site of rupture and the site away from rupture in patients with term labor and SROM. Biological processes with enrichment in the chorion included anatomic structure development, cell adhesion, signal transduction, and cell communication. The extracellular matrix–receptor interaction, graft vs host disease, and complement and coagulation cascades were among the most impacted signaling pathways by the presence of rupture in the chorion. There were no significant regional gene expression differences (rupture vs nonrupture) in the amnion transcriptome. Stereotypic impacted pathways and biological processes with enrichment were represented in the comparisons between amnion and chorion in the rupture and nonrupture sites.

This study describes, for the first time, the transcriptome of the amnion and chorion at the site of SROM and at the site distal to the site of rupture in patients with spontaneous labor at term. Previous investigations about gene expression,^45–47 protein immunolocalization and quantification,^{4,12,13,45–50} and enzymatic activity⁴ at the site of rupture have used a targeted approach. The development of high-throughput technology and computational biology makes possible the use of high-dimensional biological techniques to characterize the global changes that are associated with a particular phenomenon.^26,28 This is the first study to use transcriptomics to identify changes in gene expression in amnion and chorion at the site of membrane rupture.

Malak, Bell, McLaren, McParland, and Taylor made classic contributions by demonstrating that the fetal membranes overlying the cervix undergo morphologic^2,10,12 and biochemical^4,12,48 changes before labor. They referred to this area as the ZAM.¹⁰ The morphologic changes include decreased thickness of the trophoblast and decidua and increased thickness of the connective tissue of amnion and chorion.^2,10,12,13 The biochemical changes that were observed in this area included an increase in pro–matrix metalloproteinase (MMP)-9 (92 kd) protein concentration and enzymatic activity determined by enzyme-linked immunosorbent assay and zymography, respectively; no significant regional differences of pro–MMP-2 (72 kd) activity were detected in prelabor specimens.⁴ In the same study, increased MMP-9 protein concentration was found in specimens that were obtained after labor. Subsequently, El Khwad et al¹³ reported a zone of weakness on biophysical testing; this weak zone had increased MMP-9 and decreased tissue metallopeptidase inhibitor-3 (TIMP-3) protein by Western blot analysis. This study found that TIMP-1 was barely detectable in the membranes; TIMP-2 and TIMP-4 were detectable but did not change as a function of membrane strength. Moreover, another study from the same group demonstrated that the incubation of membranes in vitro with interleukin (IL)-1β and tumor necrosis factor alpha (TNFα) was able to induce the biophysical changes, increase MMP-9 protein (both 92 kd and 83 kd bands), and decrease TIMP-3.²⁵ These observations are consistent with an increase in amniotic fluid MMPs (MMP-1, MMP-3, MMP-7, MMP-8, MMP-9),^{19,20,22–24,51–56} neutrophil elastase,⁵⁷ IL-1β, and TNFα^{18,20,;58–61} in spontaneous labor at term and/or preterm PROM.

In a study of chorioamniotic membranes from patients with SROM or artificial rupture of membranes who delivered vaginally at term, El Khwad et al⁴⁹ used topographic mapping to identify a 10-cm (diameter) zone of weakness that surrounded the site of rupture where a decreased rupture threshold was observed. Moreover, Strohl et al¹⁴ recently demonstrated the increased separation and decreased adherence of the amnion and choriodecidua in specimens that were obtained from term deliveries, when compared with preterm membranes, which was an effect that was more pronounced in specimens from women in labor.⁶² However, there is a paucity of evidence about whether the gene expression profile of the chorion and amnion is different at the site of rupture or away from the site of rupture.

One study has examined the change in the transcriptome of the chorioamniotic membranes that were obtained from women not in labor at term and in women at term.²⁹ Importantly, membranes of women with subclinical inflammation (histologic chorioamnionitis) were excluded. The key finding of the study was that labor was associated with up-regulation of genes that are involved in the inflammatory response. These findings, which were derived originally from microarray studies, were confirmed with quantitative PCR. Therefore, spontaneous labor is associated with a signature gene expression that is consistent with inflammation of the chorioamniotic membranes, even in the absence of histopathologic evidence of such processes. Targeted studies have yielded findings that are consistent with these observations. For example, Osman et al⁴⁷ reported a higher mRNA expression for IL-1β, −6, and −8 in women in labor than in women not in labor. Such findings are consistent with previous observations that the concentrations of these cytokines are increased in the amniotic fluid of women in labor (term and preterm). ^{18,58,59,61,63–71} Of interest, Osman et al did not find regional differences in cytokine expression in the ZAM compared with specimens of membranes that were obtained distal to this site.

Our microarray results demonstrated higher expression of IL-6 and IL-8 in the chorion at the site of rupture than in the chorion from the nonrupture site. These results were confirmed by qRT-PCR for IL-6 (13.2-fold increase). The difference in study design may explain the different results between these studies: Osman et al⁴⁷ examined specimens from the ZAM in women (n =8) in labor with a cervical dilation that ranged from 4–9 cm who underwent cesarean delivery for fetal distress (no information was provided regarding the rupture status or mode of membrane rupture at the time of cesarean delivery). In contrast, the specimens that were used in our study were collected from 20 patients with confirmed SROM who underwent successful spontaneous vaginal delivery.

It is possible that the inflammatory changes at the site of rupture are due to exposure to vaginal fluid and its content, which includes microbial products, microorganisms, and inflammatory mediators. Microbial invasion of the amniotic cavity is identified with standard culture techniques in 18% of patients at term in labor with intact membranes⁷² and in 30% of patients with PROM at term.⁷³ Because bacteria can cross the chorioamniotic membranes,⁷⁴ it has been proposed that the source of the organisms is from ascending microbial invasion from the vagina. In the absence of microbial invasion of the amniotic cavity, fetal membranes that are exposed to the vagina have been thought to respond to stimuli that is present in the vaginal fluid with the production of proinflammatory cytokines and prostaglandins.⁷⁵ Concentrations of IL-1β secreted by regional decidua and the prostaglandins PGE2 and PGF2α were higher in cervical/vaginal lavage specimens from women in labor than that of women not in labor.⁷⁶ Moreover, in vitro studies with fetal membranes demonstrate reduction of elasticity and bursting tension after exposure to bacteria and products of activated neutrophils.⁷⁷

The changes in the gene expression at the rupture site may be associated with alterations in the concentration of inflammatory mediators in the amniotic fluid adjacent to the site of impending rupture. In humans, amniotic fluid prostaglandin concentrations increase before the onset of spontaneous term labor and may play a role in the initiation of labor.^78–84 Moreover, rupture of membranes at term is accompanied by increased concentrations of prostaglandins in amniotic fluid in patients with and without labor, when compared with women with intact membranes without labor.⁸⁵ The concentrations of eicosanoids, protein-degrading enzymes, and chemokines, which include tissue plasminogen activator, urokinase plasminogen activator, plasminogen activator inhibitor-2,⁸⁶ MMP-9,²¹ MMP-9/TIMP-1 ratios,²¹ and the prostaglandins PGF2α, PGFM, and PGE2,^87,88 are higher in the fluid in direct contact with the ZAM (fore-water) than in fluid from the upper compartment (hindwater) in women with term labor. The changes of the concentrations of serine proteases (tissue plasminogen activator and urokinase plasminogen activator) were thought to be a result of exposure to cytokines that are present in the vagina. Moreover, in the case of prostaglandins, the concentrations increase with advancing cervical dilation.⁸⁸ Further evidence of topographic differences in amniotic fluid markers is the increased concentration of IL-6 concentration in the forewater than in the hindwater in both term⁸⁹ and preterm¹⁸ labor. The source for the higher regional concentrations of prostaglandins and IL-6 in the forewater is unclear but may also be related to exposure to cervical and vaginal stimuli or a result of increased production by regional fetal membranes or decidua.

The results of the microarray reported herein support the concept that the fetal membranes at the site of rupture have a different pattern of gene expression than the chorioamniotic membranes away from the site of rupture. The expression of prostaglandin endoperoxidase synthase 2 gene (PTGS2), also known as cyclooxygenase 2 (COX2), was also increased in chorion from the site of membrane rupture when compared with chorion from the site away from rupture (validated by qRT-PCR). PTGS2 may be regulated by nuclear factor kappa-B (NF-κB) and is produced predominantly in amnion at term^90–92 and significantly increased in labor.⁹³ Our microarray results do not demonstrate increased PTGS2 expression in the amnion at the site of rupture when compared with amnion at the site away from rupture; however, the increase in the chorion at the site of rupture suggests that the prostaglandin biosynthesis pathway may be subject to a spatial regulation (amnion vs chorion).

ZAM is associated with a reduction in the cellularity of the cytotrophoblast and decidual layers. Apoptotic bodies (detected by electron microscopy) are increased in all regions of the fetal membranes, but specifically in the ZAM rather than in the distal regions of the membranes.⁴⁸ In addition, there was no substantial immunoreactivity for B-cell lymphoma protein 2 (BCL2), an anti-apoptotic protein, but strong for BCL2-associated X protein, a proapoptotic protein, in the decidual layer. This is consistent with observations that apoptosis occurs in the rat amnion.^94,95

Further evidence in support of this concept is the observation that there is increased cleaved caspase-3 and caspase-9 (apoptotic markers) and decreased BCL2 in the ZAM.⁵⁰ Immunohistochemical staining of BCL2 in earlier work by McLaren et al. did not demonstrate a significant difference in BCL2 expression between the ZAM and a site distal to this site.⁴⁸ Interestingly, BCL2 was one of the most significant differentially expressed genes in our microarray experiments and was decreased in the chorion at the site of rupture, compared with the nonruptured site. Further studies are required to validate these findings at the mRNA and protein levels.

As discussed earlier, there is extensive evidence of extracellular matrix remodeling at the site of rupture that is demonstrated by the increased expression of MMP-9 in the membranes before spontaneous labor. We did not find a significant difference in MMP-9 mRNA expression at the site of rupture in either the chorion or amnion, when compared with the nonrupture site. The difference in these findings may be due to the different assays. Microarrays assess changes in mRNA expression; immunoassays detect protein, and zymography detects enzymatic activity.

The PAEP gene encodes for glycodelin, which previously was also known as progestagen-associated endometrial protein or placental protein-14, which is a major progesterone-regulated lipocalin that has high carbohydrate content.^96–98 Three differentially glycosylated isoforms of glycodelin are expressed in different tissues. Glycode-lin-A is the most abundant glycoprotein in the uterus in early pregnancy. It has angiogenic,⁹⁹ immunosuppressive,¹⁰⁰ and paracrine effects on gestational tissues and has been detected in human serum,^101,102 amniotic fluid,^{101,103–105} endometrium (mainly secretory),¹⁰⁶ decidua,¹⁰⁷ and cervix.¹⁰⁸ Glycodelin-A concentrations in maternal serum and decidua increase from conception to 6–10 weeks and then decrease with advancing gestational age.¹⁰¹ Moreover, glycodelin-A concentrations in amniotic fluid are detected at 10 weeks, peak at 15–16 weeks, and decline with advancing gestational age.^101,104,105

Perturbations of glycodelin-A expression have been identified in abnormal pregnancies. It has been found to be increased in the decidua of molar pregnancies¹⁰⁹ and decreased in decidua from pregnancies that are complicated by spontaneous abortion,¹⁰⁹ ectopic implantation,¹¹⁰ intrauterine growth restriction,¹⁰⁷ and HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome.¹⁰⁷ In vitro studies have demonstrated that recombinant or immunopurified glycodelin-A induces apoptosis in monocytes and inhibits their NF-κB activation and inhibits T-cell proliferation and IL-2 secretion, which are roles that support immunotolerance during pregnancy.^100,111 Moreover, glycodelin-A suppresses MMP-2 mRNA and protein expression and MMP-2, MMP-9, and urokinase plasminogen activator enzyme activity in a first-trimester extravillous cytotropho-blast cell line.¹¹²

We report the decreased expression of PAEP in microarray experiments (3.9-fold) in the chorion at the rupture site, which was confirmed with qRT-PCR (31-fold). It is possible that the decreased PAEP expression is associated with the release of inhibition of the NF-κB signaling pathway or MMP activity. Indeed, increases in NF-κB signaling proteins⁵ and MMP-9 activity^4,49 have been described previously in supracervical membranes before the onset of labor, when compared with membranes distal to the cervix.

A disintegrin and metallopeptidase with thrombospondin type-1 motif-5 (ADAMTS-5), also known as aggre-canase-2, ADAMTS11, and implantin, is a proteinase that is known to cleave aggrecan, which is a large proteoglycan that is found in the extracellular matrix of the cartilage and cervix.¹¹³ Members of the ADAM metallopeptidase with throm-bospondin motif (ADAMTS) family are secreted molecules that can interact with the extracellular matrix. The ADAMTS-5 aggrecanase is expressed in the uterus, cervix, ovary, and placenta.¹¹⁴ In mice, ADAMTS-5 mRNA expression has been detected in the decidua in the dam in the periimplantation period, although its highest expression was detected in the placenta and is thought to contribute to extracellular matrix–remodeling at the maternal-fetal interface.^115,116 The role of ADAMTS-5 in unknown in humans. In vitro studies have described that ADAMTS-5 expression is altered by cytokines; IL-1β increases and TGF-β1 decreases the expression of ADAMTS-5 in human decidual stromal cell cultures.¹¹⁶ Other members of the ADAMTS family of aggrecanases have similar functions, although the distribution varies. For instance, in the cervix after spontaneous labor and delivery at term, ADAMTS-9 (an aggrecanase that cleaves both aggrecan and versican) is up-regulated.¹¹⁷

In both the microarray and qRT-PCR analysis, ADAMTS-5 was found to have decreased expression at the rupture site chorion, when compared with the non-rupture site chorion. We propose that, at the ZAM and the subsequent site of rupture, the maintenance of the maternal-fetal interface is no longer required and that ADAMTS-5 is rendered inconsequential. In support of this view, examination of the microarray data reveals that ADAMTS-9 is also decreased significantly in the rupture site chorion. Furthermore, our results identify the activation of the graft-vs-host disease and the allograft rejection pathways, both of which are among the top 5 most impacted and significant pathways.

Microarray analysis did not reveal a significant difference in gene expression profile of the amnion samples that were obtained from the rupture site and non-rupture site. This finding was unexpected. We expected differences in gene expression in this fetal membrane based on its role in membrane integrity and previous observations in the literature.^{5,10,36,118–120}

The lack of detectable difference may be a consequence of the large variance among patients. It is also possible that the anatomic location of the amnion precludes regional differences in the amnion at the rupture site and the nonrupture site. Indeed, the thinning of the fetal membranes in the ZAM is mostly a consequence of thinning of decidual and trophoblast layers and not the amnion.^2,10 In addition, the studies that have evaluated membrane strength indicate that the weakness in fetal membranes is predominantly due to the changes that have occurred in the chorion and less because of the amnion.¹³ Recently, our group has reported that the amnion overlying the placenta is distinctly different from the reflected amnion of the extraplacental membranes.³⁶ Using microarray analysis and tissue culture and immunoblotting techniques, we have reported that, in term fetal membranes, labor-associated switching to a proinflammatory signature is localized to placental amnion and not reflected amnion. Indeed, regional perturbations of gene expression and regulation in the amnion may occur between reflected and placental amnion but may not be evident at the site of membrane rupture.

Global analyses of mRNA expression of a particular tissue in a particular condition have been useful in the understanding of the physiologic and pathologic evidence of parturition. In spontaneous term labor, the transcriptome of the chorioamniotic membranes has an acute inflammatory gene expression signature, even in the absence of histologic chorioamnionitis.²⁹ Indeed, inflammation plays an important role in normal term labor,¹²¹ as evidenced by activation of the inflammatory pathways described in the cervix,^117,122 myometrium,¹²² and fetal membranes^29,122 of women in labor. Although different genes and specific pathways are represented in each different gestational tissue type, the core response is the activation of the inflammatory pathway. The transcriptome of the chorion at the site of rupture reported herein also supports the role of inflammation in SROM. The graft-vs-host disease, complement and coagulation cascades, and allograft rejection pathways are participants in inflammation.

The inflammatory pathway is not the only pathway of interest. In this study, the most significant and impacted pathways represented by SROM were the extracellular matrix–receptor interaction pathway and other pathways that involve cell adhesion, angiogenesis, apoptosis, and immunotolerance. Two of the top 5 most impacted pathways that have been identified in the chorion are graft-vs-host disease and allograft rejection, which sustains the role of regional withdrawal of pregnancy support at the site of rupture. Moreover, the signature pathways that have been identified were both stereotypic and novel and support the notion that human parturition and SROM are complex interactions of many biological processes.

This study describes the transcriptome of a set of matched amnion and chorion from both the site of rupture and the site away from rupture. The strengths of the study are its prospective design, inclusion of a large sample number of normal pregnant women (n =20) with SROM and vaginal delivery, exclusion of maternal diseases and specimens with documented GBS colonization of the genital tract, clinical or histologic chorioamnionitis, and meconium staining. A limitation of the study is that the differential expression of some of the genes reported herein may be due to the mechanical stress and biochemical changes that are associated with the act of rupture of membranes. To characterize the genes that are expressed differentially as a result of exposure to labor processes and/or mechanical/physiologic stress of rupture of membranes, the gene expression profile of the ZAM should be investigated. Future studies of fetal membranes may include the validation of gene regulation and the processes and pathways that have been identified to be significant and were impacted in this study.

The transcriptome of fetal membranes in spontaneous rupture in term labor is characterized by region- and tissue-specific differential expression of genes that are involved in signal transduction, cell communication, the complement and coagulation cascades, and graft-vs-host disease. In accordance with previous global studies of the transcriptome of other tissues (such as the uterine cervix) after spontaneous term parturition, genes from the extracellular matrix–receptor interaction pathway and those involved in inflammation appear to be involved in parturition. These regional differences were detected in the chorion but not in the amnion. Our findings suggest that the site of rupture in chorionic membranes has a region- and tissue-specific gene expression signature in spontaneous labor at term.