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BOR - Papers in Press, published online ahead of print October 27, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.032979
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BIOLOGY OF REPRODUCTION 72, 707–719 (2005)
DOI: 10.1095/biolreprod.104.032979
© 2005 by the Society for the Study of Reproduction, Inc.

Human Myometrial Gene Expression Before and During Parturition1

Jon C. Havelock, Patrick Keller, Ndaya Muleba, Bobbie A. Mayhew, Brian M. Casey, William E. Rainey, and R. Ann Word2

Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Texas Southwestern Medical Center, Dallas, Texas, 75390-9032


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Identification of temporal and spatial changes in myometrial gene expression during parturition may further the understanding of the coordinated regulation of myometrial contractions during parturition. The objective of this study was to compare the gene expression profiles of human fundal myometrium from pregnant women before and after the onset of labor using a functional genomics approach, and to further characterize the spatial and temporal expression patterns of three genes believed to be important in parturition. Fundal myometrial mRNA was isolated from five women in labor and five women not in labor, and analyzed using human UniGEM-V microarrays with 9182 cDNA elements. Real-time polymerase chain reaction using myometrial RNA from pregnant women in labor or not in labor was used to examine mRNA levels for three of the genes; namely, prostaglandin-endoperoxide synthase 2 (PTGS2), calgranulin B (S100A9), and oxytocin receptor (OXTR). The spatial expression pattern of these genes throughout the pregnant uterus before and after labor was also determined. Immunolocalization of cyclooxygenase-2 (also known as PTGS2) and S100A9 within the uterine cervix and myometrium were analyzed by immunohistochemistry. Few genes were differentially expressed in fundal myometrial tissues at term with the onset of labor. However, there appears to be a subset of genes important in the parturition cascade. The cellular properties of S100A9, its spatial localization, and dramatic increase in cervix and myometrium of women in labor suggest that this protein may be very important in the initiation or propagation of human labor.

cervix, gene regulation, parturition, pregnancy, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In all species, parturition is characterized by coordinate changes in the structure and function of the uterus and cervix. In the myometrium, parturition is manifest as forceful, synchronous, rhythmic contractions. The transition from a state of uterine quiescence to the onset of contractions can be viewed as the result of an increased level of contractile stimulators and the loss of mechanisms that promote relaxation [1–3]. The withdrawal of progesterone that accompanies parturition in almost all animal species is an example of retreat from factors that promote uterine relaxation. Progesterone withdrawal is associated with timely increases in the expression of genes such as the oxytocin receptor, which participate in parturition [1, 3]. In women, however, progesterone levels do not decline in maternal or fetal blood before the onset of parturition [4]. Thus, genes intrinsic to the parturition process in women may be significantly different from those identified in nonprimates.

Identification of genes intrinsic to the parturition process in women has proven to be difficult. There is evidence, however, that several genes are coordinately regulated in myometrial tissues before or during parturition [5–7]. In comparing myometrial tissue samples from laboring and nonlaboring women, differential gene expression has been identified in estrogen and progesterone receptors [8], intercellular adhesion molecule-1 [9], interleukin-8 [10], 15-hydroxyprostaglandin dehydrogenase [11], cyclooxygenase-1 and -2 (COX-1 and COX-2; also known as prostaglandin-endoperoxide synthase 1 and 2, PTGS1 and PTGS2) [12], and the myometrial gap junction protein connexin-43 [13]. This list is by no means exhaustive, and many of the molecular changes resulting in myometrial tissue remodeling and myometrial contractions are not well characterized. Studies in the baboon have demonstrated differential expression of EP1, EP2, and EP3 prostaglandin receptors within regions of the myometrium during labor [14], whereas human studies have demonstrated an expression gradient of oxytocin receptors within the myometrium [15]. These studies highlight the importance of analyzing the differential spatial regulation of gene expression in myometrium.

Analysis of myometrial gene expression by a functional genomics approach has previously been undertaken in rodent [6, 7] and human [5, 6, 16] studies. Whereas these studies have identified numerous genes that may be important in the temporal regulation of labor, gene expression analysis was conducted in tissue samples from the lower uterine segment obtained at the time of cesarean delivery from women before or after the onset of labor. During uterine contractions of labor, the lower uterine segment elongates as the fetal presenting part engages and the effaced remodeled cervix is pulled up into the maternal pelvis (cervical dilation). Thus, after the onset of labor, uterine tissue samples obtained from low cervical transverse cesarean delivery incisions contain increased amounts of remodeled cervical tissue. Changes in gene expression may reflect differences in tissue composition rather than changes in gene expression in the myometrium. Furthermore, uterine samples from the rat or mouse are invariably contaminated with endometrial epithelium, making analysis of gene expression confined to the myometrium somewhat complicated.

To better understand the spatial and temporal changes in gene expression occurring in the myometrium during parturition, we used cDNA microarrays to compare expression levels of several thousand transcripts between myometrial tissues of the uterine fundus obtained from cesarean hysterectomy specimens before or after the onset of labor in women. We found that although myometrial tissues at term exhibit few labor-related changes in genes expression at the transcriptional level, the predominant changes were associated with the inflammatory response. One of these genes, S100A9 (migration inhibitory factor-related protein-14— MRP14—or calgranulin B), belongs to the S100 family of calcium-binding proteins associated with myeloid cell differentiation, and is highly expressed in neutrophils, keratinocytes, infiltrating tissue macrophages, and epithelial cells in active inflammatory disease [17]. Herein, we compared the spatial and temporal changes in expression of S100A9 in the pregnant uterus with those of two genes have been implicated in the initiation of labor in multiple species (oxytocin receptor [OXTR] and COX-2).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Acquisition

Cervical stroma, endocervical epithelium, lower uterine segment, and fundal myometrial tissues were dissected from cesarean hysterectomy specimens and fundal myometrium was obtained from additional cesarean delivery specimens for cDNA microarray analysis (Table 1) or real-time reverse transcriptase-polymerase chain reaction (RT-PCR; Table 2). Uterine specimens were obtained immediately in the operating suite, placed on ice, and dissected within 30 min of surgery. Tissues were minced in RNAlater (Ambion, Inc., Austin, TX), snap-frozen in liquid nitrogen, and stored at –80°C until RNA isolation. Sites of placental attachment were noted. All specimens were obtained remote from identified pathology. Lower uterine segment was defined as the region between the internal cervical os and penetration of the uterine artery. Endocervical mucosa was scraped from the endocervical canal using a scalpel. Cervical stroma was obtained from the outer circumference of the cervix, avoiding endocervical glands. Squamous epithelium of the exocervix was not obtained for RNA studies. Fundal myometrium was obtained from six women undergoing vertical hysterotomy for various obstetrical indications (Table 2). All tissues were obtained in accordance with the Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas and with informed consent from women undergoing surgery. Although myometrial samples were carefully dissected by experienced investigators to minimize decidual inclusion, the possibility of residual decidual tissue as a contaminant in myometrial gene expression comparisons cannot be excluded. Additionally, populations of trophoblastic cells exist within the myometrium, including deep myometrial invasion into the arterial vasculature.


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  		TABLE 1. Clinical characteristics of women from whom myometrial samples were obtained for microarray analysis


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  		TABLE 2. Clinical characteristics of women from whom samples were obtained for analysis of gene expression using real-time PCR

RNA Preparation

Tissues were pulverized in liquid nitrogen and then homogenized in guanidinium isothiocyanate. Total RNA was isolated from cervical stroma, endocervical epithelium, lower uterine segment, and fundal myometrium as previously described [18]. Concentration of RNA was measured and purity confirmed by spectroscopy. Some total RNA was used to create polyA+ enriched samples using an mRNA purification kit (Pharmacia LKB Biotechnology Inc., Piscataway NJ). Microarray analysis was conducted both on pools of polyA+ RNA samples (n = 4 for myometrium in labor and n = 4 for myometrium not in labor) and on individual patients.

Microarray Hybridization

Differential hybridization of nonlaboring and laboring samples to microarrays representing 9182 cDNA elements were determined. Complementary DNA probe synthesis from polyA+ RNA, hybridization with the UniGEM V microarray Version 2.0, and signal analysis were conducted by Incyte Genomics (St. Louis, MO) as previously described [19, 20]. The DNA microarray for Version 2 consisted of 9182 unique probes arrayed onto glass slides. Additionally, internal control probes were included on the microarray to assess the quality of probe generation and hybridization. Isolated polyA+ mRNA (200 ng) from laboring and nonlaboring myometrium obtained from the uterine fundus was reverse-transcribed and labeled with one of two different fluorescent dyes (Cy5, red for not in labor; Cy3, green for in labor) (Operon Technologies, Alameda, CA). The two fluorescently labeled samples were simultaneously applied to each array. Following incubation the microarray was rinsed and scanned using 532 nM for Cy3 and then at 635 nM for Cy5. A signal intensity of greater than 2.5-fold over background was used for data analysis. The ratio of the two fluorescent intensities was used as a quantitative measurement of the relative gene expression between the two tissue samples.

Reverse Transcription

Reverse transcription reactions were conducted with 2 µg total RNA in a reaction volume of 20 µl. Each reaction contained 10 mM dithiothreitol, 0.5 mM deoxynucleotide triphosphates, 0.015 µg/µl random primers, 40 U RNase inhibitor (10777-019; Invitrogen, Carlsbad, CA), and 200 U reverse transcriptase (18064-014; Invitrogen). Reaction conditions were 10 min at 23°C, 60 min at 42°C, and 70°C for 15 min.

Real-Time Polymerase Chain Reaction

To ensure accurate quantification of gene expression within a given tissue type and across various tissues, quantitative real-time polymerase chain reaction (PCR) was used. Primer and probe sequences for amplifications were chosen using published cDNA sequences and the Primer Express program (Applied Biosystems, Foster City, CA) (Table 3). Where possible (i.e., OXTR and PTGS2), primers were chosen so that the resulting amplicons would cross an exon junction, thereby eliminating false positive signals from genomic DNA contamination. Taqman probes were used for detection of S100A9 and COX-2 amplicons. SYBR Green was used for OXTR amplicon detection. Gene expression was normalized to expression of 18S rRNA (4310893E; Applied Biosystems). All primer sets were tested to ensure that efficiency of amplification over a wide range of template concentrations was equivalent to that of 18S. PCR reactions were carried out in the ABI Prism 7000 sequence detection system (Applied Biosystems). The reverse transcription product from 50 ng RNA was used as template for OXTR, S100A9, and COX-2, and 5 ng RNA was used for 18S amplification. Reaction volumes were 30 µl containing 1x Master Mix (4304437 for Taqman and 4309155 for SYBR Green; Applied Biosystems). Primer concentrations were 900 nM. Cycling conditions were 2 min at 50°C, followed by 10 min at 95°C; then 40 cycles of 15 sec at 95°C and 1 min at 60°C. When SYBR Green was used, a preprogrammed dissociation protocol was used after amplification to ensure that all samples exhibited a single amplicon. Levels of mRNA were determined using the ddCt method (Applied Biosystems) and expressed relative to an external calibrator present on each plate.


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  		TABLE 3. Sequences of primers and probes used for quantification of gene expression by real time PCR

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissues were sectioned at 5 µm and mounted on the same slides as a multisample sandwich-block containing positive and negative control sections as previously described [21]. After drying, the slides were deparaffinized in xylene and rehydrated in graded alcohols to distilled water. Endogenous peroxidase activity was quenched for 10 min at room temperature using 0.3% H2O2 with 0.1% sodium azide. Slides were subjected to steam-heat epitope retrieval in EDTA buffer (1 mM pH 8.0) for 30 min. After rinsing in PBS buffer, slides were incubated in primary antibody (S100A9 Clone S 36.48; BMA Biomedicals, Augst, Switzerland, 1:200; COX-2, Neomarkers, Fremont, CA, 1:150) for 30 min at 25°C using gentle orbital rotation. Negative control specimens were processed simultaneously in an identical fashion, with the exception that PBS was used in place of primary antibody. After another rinse in PBS, slides were incubated with appropriate horseradish peroxidase-conjugated polymer (PowerVision reagent; ImmunoVision Technologies Co., Daly City, CA) was performed for 30 min at 25°C. Finally, the slides were immersed for 5 min in 25°C diaminobenzidine (Invitrogen, Carlsbad, CA), enhanced with 0.5% copper sulfate in PBS for 5 min at 25°C, counterstained in hematoxylin, dehydrated in graded alcohols, cleared in xylene, and coverslipped.

Statistical Analysis

Differences in gene expression between specimens not in labor and in labor were determined using the Student t-test for normally distributed data (S100A9 and OXTR) or the Mann-Whitney U-test for data that were not normally distributed (COX-2). Differences in gene expression among various regions within the uterus were determined using the Kruskal Wallis one-way analysis of variance on ranks using a Dunn pairwise comparison against controls.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gene Expression Profiles of Myometrium from Pregnant Women at Term Before or After the Onset of Labor

RNA isolated from a total of four samples of nonlaboring myometrium and four samples of laboring myometrium were pooled and analyzed using a genomic expression microarray (GEM). A second cDNA microarray analysis using RNA isolated from a single sample in each group was also conducted. In all, 89% of the genes examined were expressed at a level ≥2.5-fold that of background signal. Plots of all the gene comparisons from the array analyses are displayed in Figure 1. Transcripts differentially expressed ≥3-fold in laboring myometrium in the individual arrays are represented by red dots. The vast majority of genes tested were expressed to a similar degree in nonlaboring and laboring myometrium.



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  		FIG. 1. Microarray analysis comparing mRNA expression patterns in human nonlaboring and laboring myometrium. Each data point represents signal intensity of fluorescently labeled cDNA transcripts hybridizing with a total of 9182 unique genes placed on this Incyte microarray. The X and Y axes represent corrected signal intensities for Cy5 (nonlaboring) and Cy3 (laboring) after hybridization. Lines indicate the points where the fold difference in expression between the transcripts was at least 3-fold. The majority of transcripts (black circles) were similar between the two tissues. Genes designated by the red circles differed by at least 3-fold in either cDNA microarray analysis. Three transcripts were further studied using real-time PCR and immunohistochemistry. *Cyclooxygenase-2 (COX-2); {block} S100A9 (calgranulin B); {blacktriangleup} oxytocin receptor (OXTR)

From the microarray analysis, 42 transcripts (0.5%) were differentially expressed ≥3.0-fold in at least one microarray in myometrium from women in labor, and three transcripts (0.04%) were differentially expressed ≥3.0-fold in both microarrays (Table 4). In contrast, only two transcripts (0.03%) were differentially expressed ≤3.0-fold in at least one microarray in myometrium from laboring patients, and no transcripts were differentially expressed ≤3.0-fold in both microarrays. The three genes up-regulated at least 3.0-fold in both arrays were immediate early response 3 (IER3; 4.1-fold), PTGS2 (or COX-2; 4.0-fold), and FBJ murine osteosarcoma viral oncogene homolog B (FOSB; 3.8-fold).


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  		TABLE 4. Genes up-regulated ≥ 3.0-fold in laboring myometrium in either cDNA microarray comparison

Analysis of Gene Expression in Individual Myometrial Tissues

To confirm that the microarray data accurately represented differentially expressed genes in human myometrial tissues in labor compared with tissues from pregnant women not in labor, we compared mRNA expression of selected genes in multiple individual samples of myometrium from nonlaboring and laboring pregnant women using quantitative real-time RT-PCR in myometrial tissues obtained at the time of cesarean hysterectomy and vertical hysterotomy (Table 2). Initially, we found that the transcription factors NGFI-B (NR4A1) and early growth response 1 (EGR-1), two early response genes induced rapidly but transiently in response to a variety of cell stimuli, were up-regulated in myometrium from cesarean hysterectomy specimens relative to specimens obtained from vertical hysterotomy, regardless of labor status (25- and 9-fold, respectively, data not shown). These genes are transiently expressed in response to surgical trauma, blood loss, and tissue injury. Thus, we focused further evaluation on S100A9, a new parturition-associated gene identified by microarray analysis, and two genes previously described as playing important roles in the uterus during parturition, COX-2 and OXTR. By microarray analysis, myometrial COX-2 and S100A9 were shown to be increased 3-fold in labor, whereas OXTR expression was not increased significantly. A substantial number of uterine specimens were obtained from women with chorioamnionitis (determined by clinical criteria and by pathologic examination of placenta and fetal membranes). These specimens were analyzed separately as indicated in Table 2 and Figure 2.



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  		FIG. 2. Expression of COX-2, S100A9, and OTR in myometrial tissues obtained from the uterine fundus in pregnant women at term. Myometrial tissues were obtained from the uterine fundus in women undergoing cesarean hysterectomy (C-Hyst) before the onset of labor (NIL, n = 9), after the onset of normal labor (IL, n = 8–10), in labor complicated by chorioamnionitis (IL-CA, n = 5–8) or undergoing classical cesarean delivery (C-Section, NIL, n = 3; IL, n = 3). Expression of COX-2 (A), OXTR (B), and S100A9 (C) mRNA was determined by real-time PCR and expressed relative to 18S and an external standard. *P ≤ 0.01 compared with C-Hyst NIL; **P ≤ 0.01 compared with NIL C-Hyst or NIL C-Section

Compared with myometrial tissues obtained at the time of cesarean delivery, COX-2 mRNA was significantly increased in fundal myometrium obtained at the time of cesarean hysterectomy regardless of labor status. This finding suggests that either the clinical indication for cesarean hysterectomy or the procedure itself led to increased COX-2 mRNA. Nevertheless, COX-2 mRNA was not significantly different in fundal myometrial specimens from laboring and nonlaboring women whether obtained at the time of classical cesarean delivery or cesarean hysterectomy (Fig. 2). COX-2 mRNA was increased dramatically in 4 of 14 women in labor (>2 standard deviations from the mean of not-in-labor). Three of these women had chorioamnionitis.

Similar to the cDNA microarray results, OXTR mRNA levels did not vary between fundal myometrial specimens obtained from women before or after the onset of labor, and this did not vary with cesarean delivery or cesarean hysterectomy specimens or infection status (Fig. 2B). S100A9, a myeloid-related protein expressed in activated neutrophils and differentiated macrophages, was also analyzed by real-time PCR (Fig. 2C). S100A9 expression was increased significantly in myometrial specimens obtained from women in labor. Furthermore, S100A9 mRNA was increased dramatically (5-fold) in myometrial tissues obtained from women in labor complicated by chorioamnionitis compared with women not in labor (P < 0.0001). Although S100A9 mRNA was increased in laboring myometrium obtained at the time of classical cesarean delivery, the number of specimens was too small to obtain statistical significance (Fig. 2C). These data suggest that S100A9 gene expression is increased in the uterine fundus during normal labor and that, although COX-2 gene expression is not increased in the uterine fundus under physiological conditions, COX-2 mRNA may be increased significantly in the uterine fundus under pathological conditions.

Spatial Organization of Gene Expression Profiles in the Pregnant Uterus

We further characterized the spatial gene expression patterns of COX-2, OXTR, and S100A9 in the uterus from pregnant women (Fig. 3). COX-2 mRNA was increased significantly in the endocervical mucosa relative to other regions of the pregnant uterus with much lower levels in the uterine fundus. COX-2 mRNA levels were similar in the endocervix from women before and after the onset of labor. In contrast, COX-2 gene expression was increased significantly in cervical stroma of pregnant women in labor (P < 0.02). Although up-regulated in the cervix, COX-2 gene expression was not increased significantly in the lower uterine segment or uterine fundus from women in labor. The spatial pattern of S100A9 gene expression in the pregnant uterus was similar to that of COX-2, because S100A9 mRNA was increased in endocervical tissues relative to other tissue types in the uterus (Fig. 3B). However, unlike COX-2, S100A9 gene expression was increased significantly in endocervical tissue from women in labor compared with endocervix from women not in labor. In fact, S100A9 mRNA was increased dramatically in all tissue components of the pregnant uterus from women in labor. For example, in cervical stroma, S100A9 gene expression was increased 9-fold (Fig. 3B).



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  		FIG. 3. Expression of COX-2, S100A9, and OTR in myometrial tissues obtained from multiple sites in the uterus in pregnant women at term. Myometrial tissues were obtained from the endocervix (EndoCx Mucosa), cervical stroma (CX Stroma), lower uterine segment (LUS), and uterine fundus (Fundus) in women undergoing cesarean hysterectomy (C-Hyst) before the onset of labor (n = 9–12), or after the onset of normal labor (n = 8–10). Expression of COX-2 (A), S100A9 (B), and OXTR (C) mRNA was determined by real-time PCR and expressed relative to 18S and an external standard. aP ≤ 0.05 compared with LUS and Fundus; bP ≤ 0.05 compared with other tissue compartments; cP ≤ 0.05 compared with corresponding tissue not in labor; {dagger}P = 0.06 compared with corresponding tissue not in labor

The expression pattern of the oxytocin receptor in the pregnant uterus was distinct from that of S100A9 and COX-2 (Fig. 3C). Before labor, OXTR mRNA levels were increased >300-, 23-, and 4-fold in the uterine fundus compared with endocervical mucosa, cervical stroma, and lower uterine segment, respectively. Although OXTR mRNA was not increased significantly in the uterine fundus in labor compared with the myometrium before labor, OXTR mRNA was increased in cervical stroma and lower uterine segment from women in labor, but these differences did not reach statistical significance (P = 0.06).

Taken together, these data indicate that the most dramatic labor-associated changes in S100A9, COX-2, and OXTR gene expression occur in the cervix and lower uterine segment, with less pronounced changes in the uterine fundus. Expression of COX-2, S100A9, and OXTR were increased 5.7-, 9-, and 3.6-fold, respectively, in cervical stroma from women in labor. In contrast, COX-2 and OXTR were not increased significantly in the fundus in labor and S100A9 was increased only 3.6-fold. COX-2 and S100A9 mRNA are predominantly localized in the cervix, whereas OXTR is differentially expressed in the uterine fundus.

Immunolocalization of COX-2 in the Pregnant Uterus

Immunolocalization of COX-2 was conducted using a high-affinity, COX-2-specific rabbit monoclonal antibody. In myometrium at term before labor, COX-2 expression varied. In some specimens, expression of COX-2 was limited to the cytoplasm of myometrial smooth muscle cells, but the intensity of staining was weak except a few sporadic myocytes (Fig. 4A). In the majority of myometrial fundal tissue from cesarean hysterectomy specimens before labor, however, cytoplasmic and perinuclear immunostaining was found in most myocytes with no staining in vascular smooth muscle cells (Fig. 4B). This pattern was not different from that of specimens obtained after the onset of labor (Fig. 4C). In myometrial specimens obtained from women with chorioamnionitis, staining for COX-2 was also observed in fibroblasts between smooth muscle bundles and tissue macrophages (Fig. 4D).



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  		FIG. 4. Immunolocalization of COX-2 in human fundal myometrium. Myometrial tissues were obtained at the time of cesarean hysterectomy from women at term before the onset of labor (A and B), after the onset of labor (C), and in labor complicated by chorioamnionitis (D). Before the onset of labor, COX-2 was either localized to a few sporadic myocytes (A) or to numerous myometrial smooth muscle cells throughout the tissue section (B). This pattern of COX-2 expression was indistinguishable from that of women in labor (C). In myometrial tissues from women with chorioamnionitis, COX-2 was also localized to myocytes, stromal fibroblasts, and tissue macrophages (arrows). Bar = 40 µm

In cervical tissues from women before labor, COX-2 was highly expressed in endocervical epithelial cells, but cells throughout the cervical stroma were either negative or demonstrated weak staining (Fig. 5, A and C). In cervical tissues obtained after the onset of labor, COX-2-positive cells were localized not only in endocervical epithelial cells, but also stromal fibroblasts, cervical smooth muscle cells, occasional tissue macrophages, and endothelial cells lining small arterioles (but not venules) (Fig. 5, B and D). COX-2 staining was absent in infiltrating leukocytes and vascular smooth muscle (Fig. 5).



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  		FIG. 5. Localization of COX-2 expression in human cervical tissues during pregnancy. Immunohistochemistry was used to localize expression of COX-2 in tissue sections from women at term before (A and C) or after the onset of labor (B and D). Sections from the endocervical mucosa are represented in (A and B), whereas sections from the deeper cervical stroma are seen in (C and D). epi, Epithelial cells lining endocervical crypts; str, cervical stroma; l epi, luminal epithelial cells lining the endocervical canal; sq, squamous epithelium. Magnification x200

The finding that COX-2 expression was highly localized to endocervical epithelial cells in pregnant women at term, even before the onset of labor, led us to evaluate the expression of COX-2 in cervical tissues obtained from nonpregnant women and pregnant women earlier in gestation (Fig. 6). Of interest, epithelial cells lining the lumen of the endocervical canal were COX-2-positive in all tissues examined, but the intensity of immunostaining in these cells in the nonpregnant cervix or cervix in early gestation appeared to be less than that seen in term pregnancy, and staining was absent in a subset of luminal epithelial cells (Fig. 6, A–C; compare with Fig. 5, A and B). In nonpregnant women, COX-2 immunoreactivity was less pronounced in highly differentiated, mucous-secreting cells that line the endocervical crypts compared with luminal epithelial cells (Fig. 6A). In tissues obtained at 13 and 28 wk of gestation, COX-2 staining intensity increased in epithelial cells of the crypts (Fig. 6, B and C). The results suggest that although COX-2 may be constitutively present in endocervical epithelial cells, the level of its expression is regulated throughout pregnancy.



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  		FIG. 6. Localization of cervical COX-2 in nonpregnant women and pregnant women during early pregnancy. Immunohistochemistry was used to localize expression of COX-2 in tissue sections from nonpregnant (A), first trimester pregnancy (B), and second trimester pregnancy (C). Note prominent staining in luminal epithelial cells relative to cells lining the endocervical crypts in nonpregnant cervix (A). Staining of cells lining the crypts was more prominent as pregnancy progressed (B and C). epi, Epithelial cells lining endocervical crypts; str, cervical stroma; L epi, luminal epithelial cells lining the endocervical canal. Data are representative of three specimens obtained from nonpregnant women and three specimens from pregnant women in early pregnancy (not in labor). Magnification x200

Immunolocalization of S100A9 in Myometrium and Cervix

The two Ca2+-binding proteins, S100A8 (calgranulin A, 11 kDa) and S100A9 (calgranulin B, 14 kDa) heterodimerize to form a protein complex that is highly expressed in tissues involved in active inflammatory disease [17]. Utilizing an antibody that recognizes S100A9 alone as well as bound in its heterodimeric complex, the protein was localized to cervical and myometrial neutrophils and, to a lesser extent, tissue macrophages. In cervical tissues from women before labor, staining was limited to intravascular granulocytes with rare reactive cells in the stroma (Fig. 7A). Likewise, in myometrium before labor, rare reactive cells were noted in myometrial smooth muscle bundles or intervening stromal cells (Fig. 7D). The number of immunoreactive cells was increased dramatically in cervical and myometrial tissues in labor (Fig. 7, B and E). In addition, in tissues from women in labor, S100A9 was found in vascular endothelium adjacent to marginating neutrophils and monocytes, suggesting that secretion of the complex onto vessel walls is involved in the migration of leukocytes into the cervical stroma. In the laboring cervix, S100A9-positive cells within the venules were adherent to endothelial cells and extravasation of numerous S100A9-positive leukocytes was noted in the cervical stroma and myometrium. Thus in myometrium and cervical stroma in labor, S100A9-positive leukocytes were no longer limited to intravascular neutrophils, but were also found adherent to the cervical stroma and to the stroma between myometrial fascicles (Fig. 7, B and E). In cervical and myometrial tissues from women in labor with chorioamnionitis, the number of infiltrating S100A9 leukocytes increased dramatically (Fig. 7, C and F). Immunostaining for S100A9 in cervical, lower uterine segment, and myometrial tissues from the same uterine specimens revealed that the intensity of staining and number of immunoreactive cells increased progressively in sections taken from the uterine fundus to the cervix and endocervical mucosa (not shown).



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  		FIG. 7. Expression of S100A9 in cervical (A–C) and myometrial (D–F) tissues from pregnant women before (A and D) and after (B, C, E, and F) the onset of labor. Sections of cervical and myometrial tissues obtained at the time of cesarean hysterectomy were reacted with S100A9 antibody. S100A9 was localized to cervical and myometrial neutrophils and, to a lesser extent, to tissue macrophages. In tissues from women in labor (B, C, E, and F), the number of immunoreactive leukocytes was increased dramatically. Immunostaining was not present in negative control sections reacted with all reagents except primary antibody (not shown). Sections are representative of data obtained from three different tissues in each group with similar results. Magnification x100


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this investigation, we sought to identify differential gene expression in the uterine fundus during labor because gene expression patterns in the lower uterine segment may not reflect those associated with the contracting muscular component of the pregnant uterus. The finding of a paucity of genes that are differentially transcriptionally regulated in myometrium in labor compared with not in labor is consistent with findings in previous functional genomics studies using myometrium from the lower uterine segment obtained at the time of cesarean delivery [5, 6, 16, 22]. The magnitude of change in gene expression in the fundus of women in labor was less than that reported in the lower uterine segment [6], and in contrast to findings in rodent pregnancy, there were no obvious changes in genes known to be associated with smooth muscle contraction or relaxation. It should be emphasized, however, that previous studies [5, 6] used not only myometrium from the lower uterine segment, but also different gene arrays and, in the study by Bethin et al. [6], genes expressed in the myometrium in labor at term were compared with preterm myometrium. Despite these differences in technique and method of uterine sampling, comparisons of gene expression profiles of human myometrium before and after the onset of labor agree that, compared with studies conducted in murine and rat pregnancy, changes in gene expression at the transcriptional level in myometrial tissues obtained from laboring women are minimal, and the predominant changes were associated with the inflammatory response. Findings are concordant for at least five genes, all of which are involved in the inflammatory response; namely, homolog of murine myxovirus resistance 1 (MX1), interferon alpha-inducible protein (G1P3), interferon-induced transmembrane protein 1 (IFITM1; [9–27]), FcgammaRIIIA/CD16 (FCGR3A), and alpha-1-antichymotrypsin (SERPINA3).

One of the microarray comparisons demonstrated a significant increase in expression of immunoglobulin genes, with immunoglobulin kappa variable 1D-8 (IGKV1D-8) demonstrating the greatest increase in labor compared with not in labor. The finding of increased immunoglobulin gene expression in one microarray is interesting, as B-lymphocytes were sparsely identified in myometrial specimens from laboring patients [23], and lymphocytes in uterine cell suspensions from pregnant mice do not exceed 5% of total leukocytes [24]. Up-regulation of the immunoglobulin genes was confined to the array containing mRNA from a single patient in each group, but none of these genes were up-regulated in the array containing mRNA from pooled myometrial specimens. Whether or not increased expression of immunoglobulin genes in myometrium occurs during labor awaits further confirmation. Nevertheless, up-regulation of FcgammaRIIIA/CD16, multiple interferon-induced regulatory proteins, and E-selectin (an adhesion molecule involved in attracting neutrophils, monocytes, and some lymphocytes [25]) in myometrial tissues from women in labor suggests that coordinated expression of several genes involved in the inflammatory cascade in myometrium accompanies parturition in women.

Validation of Microarray Results

To validate results of the microarrays, mRNA levels were determined in numerous individual cesarean hysterectomy samples and results were compared with those obtained in fundal myometrial tissues obtained at the time of vertical hysterotomy. Myometrial tissues obtained from uterine specimens at the time of cesarean hysterectomy demonstrated increased expression of several genes involved in the acute phase response (EGR1, NR4A1, and COX-2). However, because expression of these genes was very low in fundal myometrium obtained at the time of vertical hysterotomy, up-regulation of these genes from cesarean hysterectomy specimens, even in the nonlaboring myometrium, may be due to several factors unrelated to labor status (e.g., underlying indication of cesarean hysterectomy, trauma of the surgical procedure, infusion of oxytocin during the hysterectomy, or hypoxic insult after clamping the uterine artery). Thus, these genes may not be involved in physiologic labor. In the case of S100A9 and oxytocin receptor, however, mRNA levels were similar in nonlaboring myometrium obtained at the time of vertical hysterotomy or cesarean hysterectomy, and results obtained from numerous individual tissue specimens confirmed the findings from the microarray analysis.

Oxytocin Receptor Gene Expression in Human Myometrium During Pregnancy

By cDNA microarray analysis and real-time PCR, OXTR gene expression was not up-regulated in fundal myometrium in labor. This result is consistent with some studies [26, 27], but not others [14]. OXTR gene expression was previously demonstrated to be up-regulated in labor using suppression subtractive hybridization [22]. Other studies using a functional genomics approach have revealed no significant difference in OXTR gene expression [5, 6]. Temporal changes in myometrial OXTR gene expression in the fundus during human pregnancy may be related to slow increases in uterine stretch associated with fetal growth [28], with maximal increases in gene expression occurring at term but before the active phase of labor.

The spatial pattern of myometrial OXTR gene expression in fundus and lower uterine segment is consistent with that described by others [15, 26]. We further characterized the spatial expression of this gene demonstrating significantly decreased OXTR mRNA levels in the cervix relative to the lower uterine segment and still further decreases in endocervical mucosa relative to cervical stroma. This pattern of gene expression is believed to be one mechanism for the coordinated regulation of myometrial contractions from myometrium to cervix that generates the necessary force for cervical dilatation and expulsion of the fetus. Whereas oxytocin signal transduction does not seem to be essential for initiation of labor, as evidenced by normal lengths of gestation and active phase of labor in oxytocin knockout mice [29], its role as a powerful uterotonin and use in labor induction and augmentation highlight its importance in parturition and recovery of the parturient from pregnancy. In this study, in which tissues were collected at well-defined regions of the uterus rather than surgical incision sites, we found that although oxytocin receptor gene expression was decreased in the lower uterine segment and cervix relative to the fundal myometrium, oxytocin gene expression was up-regulated in these tissues during labor (P = 0.06). These results indicate that uterine sampling techniques and variability in the location of surgical incisions during cesarean delivery may lead to discrepant results as to whether or not OXTR is increased in human myometrium during labor. OXTR gene expression was remarkably abundant in pregnant myometrium and lower uterine segment (Ct = 18–21 using 50 ng RNA). Thus, the 3-fold increase in OXTR in lower uterine segment and cervix in labor represents a dramatic increase in the number of OXTR transcripts in these tissues.

Spatial and Temporal Expression of COX-2 in the Uterus During Human Pregnancy

There is abundant evidence to support a role for prostaglandins in labor and myometrial stimulation [30]. Prostaglandins are synthesized from arachidonic acid, and are converted to prostaglandin PGH2 by prostaglandin endoperoxide H synthase (PGHS) or COX. Two isoenzymes exist, COX-1, or the constitutive form; and COX-2, the inducible isoenzyme. These enzymes are responsible for the rate-limiting step in prostaglandin biosynthesis. COX-2 is up-regulated by various growth factors and cytokines, and has been shown in some studies to be increased during parturition in the myometrium of the rat [31], mouse [32], and human [33, 34], whereas other human studies have revealed no increase in COX-2 expression [35, 36].

In agreement with others in which fundal specimens were obtained in a similar fashion at the time of cesarean delivery [11], COX-2 expression was similar in fundal myometrium from women before and after labor. Up-regulation of COX-2 expression in three of six myometrial tissues from women with chorioamnionitis, but no change in COX-2 expression in labor at the time of cesarean delivery or cesarean hysterectomy, suggests a role for COX-2 in inflammation-mediated labor and differences in regulation of normal and inflammation-mediated initiation of labor. Studies in a murine model have demonstrated induction of uterine COX-2, but not COX-1, during inflammation-mediated preterm labor caused by lipopolysaccharide (LPS) administration [32]. Furthermore, COX-1 deficient mice, which show delay in the onset of term labor, did not show a delay in onset of preterm labor after administration of LPS.

COX-2 expression has previously been shown by Western blot analysis to be spatially regulated in human myometrium in pregnancy and labor, with an increasing concentration gradient from fundus to cervix [12]. Using real-time PCR, we confirmed and extended these studies to indicate that COX-2 gene expression is also differentially regulated in endocervix mucosa compared with cervical stroma. This gradient of gene expression parallels that of leukocyte migration into the uterus during labor [23]. However, although COX-2 was localized to macrophages and a subset of monocytes within the cervix and myometrium, COX-2 expression was not restricted to these cell types. Rather, in the laboring cervix, COX-2 was expressed predominantly in cervical epithelial cells, activated endothelial cells, and stromal fibroblasts. In agreement with mRNA levels of COX-2, the number of COX-2-positive stromal fibroblasts per tissue cross section appeared to be increased in both myometrial and cervical tissues in labor with the number of immunoreactive cells increased in the cervix relative to myometrium. Fibroblasts cultured from human myometrium obtained from the lower uterine segment express different surface antigens with distinct cytokine and chemokine patterns of expression [37] and distinct COX expression profiles [38]. Although the uterine cervix is well known to contain more fibroblasts and less smooth muscle cells than myometrium, differences in the relative proportions of fibroblast subtypes between the two tissues may also contribute to the relatively increased expression of COX-2 in cervix compared with myometrium. In addition, it is interesting to note that in contrast to cervical stroma, COX-2 gene expression was increased in endocervical mucosa in women at term, even before the onset of labor. The data from immunohistochemistry suggest that endocervical glandular epithelial cells are responsible for up-regulation of COX-2 in the endocervix at term. Further studies will be necessary to determine the time course of changes in COX-2 gene expression in relation to cervical ripening.

Spatial and Temporal Regulation of S100A9 (Calgranulin B) in the Pregnant Uterus

S100A9 and S100A8 (calgranulin A) form heterodimeric complexes that are secreted upon stimulation with inflammatory mediators [39]. The partially antagonistic function of S100A8 and S100A9 alone on the Ca2+-dependent heterocomplex [40] gives rise to versatility in regulation of the inflammatory response. Previously, S100A8 was shown to be involved in the regulation of fetal-maternal interactions as null mutations of the mouse S100A8 gene result in embryo resorption at the maternal interface [41]. Herein, we demonstrate that S100A9-positive cells are associated with parturition and the termination of pregnancy. S100A8/A9 heterodimers specifically bind fatty acids in a calcium-dependent manner, and arachidonic acid is specifically bound to the protein complex [42]. The complex binds to the major fatty acid transporter (CD36) of endothelial cells and interacts with heparan sulfate proteoglycans via the S100A9 subunit [43]. In COX-2-positive cells in the cervix and myometrium during labor, particularly in the setting of infection, secreted complexes may facilitate arachidonic acid transport and prostaglandin production.

The signals responsible for the initiation of leukocyte recruitment during cervical ripening and parturition are unknown. S100A9 may be involved in neutrophil migration to the cervix and myometrium because S100A9 enhances monocyte adhesion to endothelial cells [44] through activation of the ß2 integrin Mac-1 on neutrophils [44]. S100A8/S100A9 heterodimers also facilitate monocyte migration through the endothelium [45]. Moreover, studies in a murine model demonstrated that S100A9 is also involved in inducing the release of neutrophils from the bone marrow [46]. Thus, S100A9 may be involved in leukocytosis that accompanies parturition. Current evidence suggests that infiltration of the myometrium by neutrophils and macrophages occurs in spontaneous labor at term [23, 47]. S100A9 may represent a new chemotactic factor contributing to macrophage and neutrophil migration into myometrium and cervix during the initiation or progression of the parturition process. The spatial gradient of S100A9 gene expression and S100A9-positive leukocytes in the uterus at term suggests that initial recruitment of these proteins may occur in the cervix before uterine contractions of labor.

Summary and Perspective

The predominant changes in gene expression in fundal myometrium in labor were genes associated with the inflammatory response. At least one of these genes has a distinct spatial pattern of expression in the pregnant uterus and appears to be associated with the onset of physiologic labor at term (i.e., S100A9). Other genes expressed in the myometrium may be associated with the pathophysiology of preterm labor or labor complicated by intrauterine infection or hypoxic insult (e.g., COX-2). Evidence exists for inflammatory cytokines in mediating parturition [48] and stimulating COX-2 gene expression. Inflammation-mediated preterm labor in the mouse resulted in increased uterine COX-2, but not COX-1, gene expression, and inhibition of COX-2 resulted in arrest of LPS-induced preterm labor [32].

Recent results from several laboratories suggest that although progesterone levels at term do not decrease in human pregnancy, functional progesterone withdrawal may occur through changes in progesterone receptor isoforms [49], progesterone receptor coactivators [50], or changes in local progesterone metabolism [51]. In the current study, changes of gene expression in the uterine fundus in labor are not associated with any known progesterone receptor-responsive genes. It should be noted that the time course of expression of these genes cannot be studied in human pregnancy. Thus, whether these genes are involved in the initiation of labor at term or a result of the labor process is unknown. Changes occurring in human myometrium associated with initiation of uterine contractions of labor may be posttranslational in nature. Posttranslational modifications may result in changes in myocyte membrane potential, increased activity of Ca2+ channels [52], or gap junction formation and permeability [53]. Alternatively, or additionally, contractions of the uterine fundus in labor may simply occur in response to factors liberated by the remodeled cervix or fetal membranes at term with no change in steady-state levels of protein or mRNA expression.


   ACKNOWLEDGMENTS
 
We thank the physicians and staff of Parkland Memorial Hospital, and Ms. Sheila Brandon and Ms. Valencia Hoffman for their valuable assistance in tissue procurement. The advice and technical expertise of Rodney Miller, M.D. (ProPath Laboratory, Inc., Dallas, TX) is gratefully acknowledged. We thank Ms. Tonja Dimio for assistance in manuscript preparation and administration of the Human Tissue and Biologic Fluid Core Laboratory.


   FOOTNOTES
 
1 Supported by grant HD11149 from the National Institutes of Health. Back

2 Correspondence: R. Ann Word, Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9032. FAX: 214 648 9242; Ruth.Word{at}UTSouthwestern.edu Back

Received: 9 June 2004.

First decision: 2 July 2004.

Accepted: 13 October 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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