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Molecular Endocrinology, doi:10.1210/me.2003-0007
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Molecular Endocrinology 17 (8): 1454-1469
Copyright © 2003 by The Endocrine Society

Microarray Analysis of Uterine Gene Expression in Mouse and Human Pregnancy

Kathleen E. Bethin, Yoshihiko Nagai, Robert Sladek, Minoru Asada, Yoel Sadovsky, Thomas J. Hudson and Louis J. Muglia

Departments of Pediatrics (K.E.B., M.A., L.J.M.), Obstetrics and Gynecology (Y.S., L.J.M.), and Molecular Biology and Pharmacology (L.J.M.), Washington University School of Medicine, St. Louis, Missouri 63110; and Montreal Genome Centre (Y.N., R.S., T.J.H.), McGill University Health Centre, Montreal, Quebec, Canada H3A 2B4

Address all correspondence and requests for reprints to: Louis J. Muglia, M.D., Ph.D., Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8208, St. Louis, Missouri 63110. E-mail: Muglia_L{at}kids.wustl.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Improved care of infants born prematurely has increased their survival. However, the incidence of preterm labor has not changed. To understand the processes involved in preterm labor, we used oligonucleotide microarrays to study gene expression in murine and human uterus during pregnancy. The induction of enzymes for prostaglandin synthesis was used as a marker for important changes during pregnancy because prostaglandins strongly contribute to both human and murine labor. We identified 504 genes that changed at least 2-fold between d 13.5 and 19.0 in the gravid mouse uterus. In the pregnant human myometrium, we found 478 genes that changed at least 2-fold in either term or preterm labor compared with preterm nonlabor specimens and 77 genes that significantly varied in both preterm and term labor. Patterns of gene regulation within functional groups comparing human preterm and term labor were similar, although the magnitude of change often varied. Surprisingly, few genes that changed significantly throughout pregnancy were the same in the mouse and human. These data suggest that functional progesterone withdrawal in human myometrium may not be the primary mechanism for labor induction, may implicate similar mechanisms for idiopathic preterm and term labor in humans, and may identify novel targets for further study.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE GENETIC PROGRAM that coordinates the timing of the onset of parturition with the rate of fetal maturation remains one of the central unsolved problems in reproductive biology. In addition to being of intrinsic scientific interest, understanding the mechanisms initiating normal and abnormal labor is of tremendous medical importance, as premature labor and delivery complicate up to 10% of human pregnancies. Although considerable improvements in promoting survival of premature infants have occurred over the past 30 yr, these infants still account for the majority of neonatal mortality and are subject to long-term cognitive and physical impairments (1, 2, 3). Interventions that could prevent spontaneous preterm labor and delivery would clearly be the optimal mechanism to improve neonatal health. Unfortunately, due to our limited understanding of the mechanisms of human labor, no change in the frequency of preterm delivery has occurred in the last several decades (3).

A major hindrance for the analysis of human labor is the limited ability to extrapolate endocrine changes important for labor in one species to those of another. As would be expected for a process as fundamental for the maintenance of a species as parturition, many conserved pathways have been identified. By focusing on these conserved pathways, fundamental insights into the initiation and progression of labor are likely to be achieved. Using targeted mutagenesis of candidate genes, critical components of the parturition cascade for labor in mice have emerged. The production of prostaglandin F2{alpha} by cyclooxygenase (COX)-1 has been identified as essential for luteolysis and the initiation of labor in mice (4, 5, 6). The ability to promote or attenuate the progression of labor in humans by administration of prostaglandins or prostaglandin synthesis inhibitors, respectively, implicates prostaglandin production as a key component of human labor as well (7, 8, 9).

Oligonucleotide microarray analysis, by allowing the simultaneous, quantitative assessment of the expression of thousands of genes, has shed insight into a wide range of developmental, oncological, and pharmacological processes (10, 11, 12). Consequently, we expect that microarray analyses will be useful for identifying changes in gene expression critical for the process of parturition. In this study, we analyzed both murine and human uterine samples using oligonucleotide microarrays to gain insight into murine uterine genes associated with the induction of prostaglandin biosynthesis at term gestation, and to identify those molecular events that could contribute to the timing of human term and preterm labor.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genome-wide changes in uterine gene expression during pregnancy were studied using Affymetrix (Santa Clara, CA) GeneChip microarrays. Our initial analyses used the mouse uterus as the source of RNA because the molecular components leading to parturition have been reasonably well characterized in this species. The critical uterine contribution in mice is clear both for prostaglandin production and requisite changes in myometrial contractility, but the fundamental controlling genes remain obscure. COX-1 catalyzes the first committed step in prostaglandin biosynthesis and has been shown to be essential for normally timed labor in the mouse (6, 13). COX-1 mRNA levels are low in the gravid murine uterus until approximately d 15.5 of gestation when levels rapidly increase and stay high until the delivery of the pups. The induction of COX-1 mRNA provides one of the earliest available markers for impending labor. Therefore, we chose to compare relative levels of gene expression at gestational d 13.5 when COX-1 expression is low, with gestational d 16.5 when COX-1 mRNA expression is high and prostaglandin levels are low, and gestational d 19.0 when COX-1 expression is high and prostaglandin levels are high, just before delivery at d 19.5.

We identified a total of 504 genes that significantly changed at least 2-fold during murine pregnancy. As initial validation of our microarray profiling, we detected the anticipated induction of COX-1 mRNA at d 16.5 of gestation and sustained elevation at d 19.0 (Fig. 1Go). To provide a further conceptual framework for identifying regulatory networks, we used a variety of clustering techniques to classify these genes based on their temporal expression profile (Fig. 1Go and information published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend. endojournals.org.). Those genes clustering with COX-1, and thus possibly regulated by the same factors as those involved in COX-1 gene expression or themselves playing a role in COX-1 gene regulation, are listed in Table 1Go (similar lists for each cluster are provided as supplemental data). To investigate expression patterns of entire functional groups of genes, we hierarchically clustered genes within GenMAPP functional groupings (Fig. 2Go). COX-1 is shown in the metabolism functional group (Fig. 2Go). In approximately half of the functional groups, similar numbers of genes increased or decreased in expression. In contrast, we found many more genes decreased in expression as opposed to increased in expression in the immune/inflammatory, protease/protease inhibitor, and cell adhesion categories (Fig. 2Go).



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  		Fig. 1. Regulatory Cluster Analysis of Relative mRNA Expression in Gravid Mouse Uterus

After normalizing the absorbance data, genes that changed expression at least 2-fold (n = 504) compared with d 13.5 were clustered into eight different groups by comparing their expression levels and regulation patterns. The x-axis for each graph is days post conception (d 13.5, 16.5, and 19.0). The y-axis of each graph is the relative mRNA expression level (absorbance intensity). The cluster containing COX-1 is indicated.

 

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  		Table 1. Genes that Cluster with COX-1

 


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  		Fig. 2. Functional Grouping of Hierarchical Clustered Mouse Genes from Gravid Uterus

The genes shown in Fig. 1Go were divided into functional groups based on GenMAPP, and relative changes in their expression level were indicated by a color code. Red indicates that the gene is up-regulated, and green indicates that the gene is down-regulated. The intensity of the color is proportional to the change in gene expression level compared with the d 13.5 baseline sample. Black indicates no change from d 13.5. The first column is d 13.5 (all in black), the second column is d 16.5, and the third column is d 19.0.

 
A characteristic feature of gene regulation during pregnancy is the coordinate modulation of factors along the same metabolic pathway or serving similar functions. For example, in addition to COX-1, prostaglandin F synthases are induced at approximately the same time during gestation, and a panel of contraction-associated proteins is simultaneously induced at term in the myometrium (14, 15, 16). One mechanism that could result in these coordinated changes is induction of specific transcription factors at defined points during gestation. Other fundamental changes evaluated as possible contributors to the maintenance of pregnancy are endometrial or myometrial proliferation genes and genes contributing to remodeling of the uteroplacental interface. Therefore, when analyzing significantly changing genes, we focused on transcription factors, cell cycle control proteins, and matrix-degrading enzymes.

Transcription Factors
Of the 504 genes induced or repressed during late murine gestation in the mouse uterus, 23 probe sets representing transcription factors were identified (Table 2Go). Elf3, a member of the Ets family of transcription factors (17), increases 5-fold from d 13.5–19.0. Northern analysis (data not shown) and in situ hybridization of murine gravid uterus confirmed this large change (Fig. 3Go). Elf3 is found exclusively in the endometrium of nongravid and gestational d 16.5 and 19.0 uterus. There is little or no expression of Elf3 in d 13.5 uterus. In addition, as shown with the data from the microarray analysis, Elf3 expression increases between d 13.5 and 16.5.


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  		Table 2. Transcription Factors, Cell Cycle Factors, and Tissue Remodeling Genes that Changed at Least 2-Fold in Mouse Uterus between Days 13.5 and 19.0

 


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  		Fig. 3. In Situ Hybridization of Elf, c-Fos, and p57Kip2

Shown are dark-field images from murine nongravid (NG), or gravid d13.5, d16.5, and d19.0, uterus (magnification, x40). The slides were counterstained with hematoxylin and eosin. Areas of hybridization appear white or silver, demonstrating deposition of silver grains. E, Endometrium; M, myometrium.

 
The Sox genes are a family of transcription regulators that are related to the SRY (testis determining) gene and contain a high mobility group box DNA-binding domain. Sox4 mRNA has previously been shown to be present in mouse uterus and is negatively regulated by estrogen and positively regulated by progesterone (18, 19, 20). Sox4 showed a 2.5-fold increase in expression on d 19.0 compared with d 13.5 by microarray analysis, and changes in signal intensity by in situ hybridization to gravid mouse uterus are consistent with this (Fig. 4Go). Sox4 expression is not detectable in the nongravid murine uterus. Myometrial expression of Sox4 is detectable on d 13.5 and is slightly increased in the myometrium on d 16.5. On d 19.0, Sox4 expression is further increased in the myometrium.



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  		Fig. 4. In Situ Hybridization of Sox4 and ST3

Shown are dark-field (rows 1 and 3) and bright-field (rows 2 and 4) images from murine nongravid (NG), or gravid d13.5, d16.5, and d19.0, uterus (magnification, x40). The slides were counterstained with hematoxylin and eosin. Areas of hybridization appear white or silver, demonstrating deposition of silver grains. E, Endometrium; M, myometrium; D, decidua.

 
The transcription factor c-Fos is a member of the immediate early gene family (21). Microarray analysis shows a 2.2-fold up-regulation of c-Fos in d 19.0 mouse uteri as well as an induction of c-Fos in myometrial samples obtained from women in labor (see below). In situ hybridization identifies c-Fos transcripts in the endometrium and not the myometrium of the nongravid uterus (Fig. 3Go). During murine pregnancy expression of c-Fos is found in the myometrium on d 13.5 and 16.5 at approximately equal levels. By d 19.0, expression in the myometrium has decreased, but c-Fos is highly expressed in the fetal membranes.

Cell Cycle Regulation Genes
We found four genes involved in cell cycle control whose expression changed significantly between gravid d 13.5 and 19.0 in the mouse uterus (Table 2Go). p57Kip2, a member of the p21CIP1 family of cyclin-dependent kinase inhibitors (22, 23), demonstrated a 2.8-fold increase in mRNA levels in d 19.0 mouse uteri compared with d 13.5. In situ hybridization demonstrated that p57Kip2 is expressed in the endometrial stroma of the nongravid uterus (Fig. 3Go). On gestational d 13.5 and 16.5, it is expressed in the myometrium at approximately equal levels. At d 19.0, the myometrial expression is significantly increased, in agreement with the data from the microarray analysis.

Matrix Remodeling Enzymes
Two genes involved in extracellular matrix reorganization changed significantly in expression in late murine gestation (Table 2Go). Matrix metalloproteinases are zinc-dependent endopeptidases that have activity against all of the components of the extracellular matrix. Stromelysin-3 (ST3), a member of the matrix metalloproteinase family (24, 25), increased 7.8-fold at d 19.0 of gestation. In situ hybridization revealed that ST3 was expressed at low levels in the nongravid and d 13.5 uterus (Fig. 4Go). ST3 expression significantly increased on d 16.5 in the decidua basalis, the uterine region in contact with the placenta. On d 19.0, ST3 expression is increased further in the decidua.

Elf3 and COX-1 Gene Regulation
The timing for induction of Elf3 relative to COX-1 and the colocalization of Elf3 and COX-1 in the endometrium, suggest that Elf3 could be involved in the induction of COX-1 gene expression found during late gestation. We analyzed the 5'end of the mouse COX-1 gene (26) and GenBank accession no. AL929054) for possible Elf3 binding sites. Using the Match 1.0 software (www.gene-regulation.com), we identified three possible Elf3 binding sites, two overlapping in intron 1 and one at the junction of intron 1 and exon 2 (Fig. 5AGo). To further determine whether Elf3 could modulate COX-1 gene expression, we cotransfected a COX-1-ß-galactosidase reporter plasmid, encompassing the region from 2.4 kb upstream of the COX-1 transcription start site through base pair +193 in exon 2, with an Elf3 expression vector (27). We found that Elf3 cotransfection specifically augmented transcription from the COX-1 promoter (Fig. 5BGo). Transfection of a COX-1 reporter construct lacking the intron 1 and exon 2 sequences encompassing the possible Elf3 binding sites, together with the Elf3 expression vector, did not lead to augmented transcription relative to the promoterless vector backbone, further implicating the possible binding sites within intron 1 as important for COX-1 gene expression (data not shown).



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  		Fig. 5. Elf3 Augments COX-1 Promoter Activity

A, Schematic diagram of the mouse COX-1 transcription promoter region from 2.4 kb upstream of the transcription initiation region through exon 2. The putative Ets-factor binding sites are shown. B, Cotransfection of a COX-1-ß-galactosidase reporter plasmid (p5'COX1-E2) with the indicated amounts of an Elf3 expression vector in Chinese hamster ovary cells results in significantly increased reporter activity in comparison to the promoterless vector (pnlacF) or p5'COX1-E2 without the added Elf3 expression vector (P < 0.05 p5'COX1-E2 vs. pnlacF at each concentration of cotransfected Elf3). Shown is fold induction above the same plasmid transfected without the Elf3 expression vector.

 
Human Myometrial Gene Expression
Our ultimate goal is to identify uterine genes that are important in human labor. We performed microarray analysis using myometrial RNA obtained during cesarean section from women who were either preterm and not in labor, or preterm and in labor, or at term and in labor. There are 478 genes that significantly change either during preterm or term labor compared with preterm nonlabor. These genes were clustered into eight groups by heuristic and K-Means clustering techniques (Fig. 6Go and supplemental data). We identified a total of 77 probe sets that changed significantly in association with both preterm and term labor (Tables 3Go and 4Go).



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  		Fig. 6. Regulatory Cluster Analysis of Relative mRNA Expression from Human Gravid Myometrium

After normalizing the absorbance data, the genes that changed at least 2-fold (n = 478) compared with preterm nonlabor, were clustered into eight different groups by comparing their expression level and regulation patterns. The y-axis of each graph is the relative mRNA expression level (absorbance intensity). The x-axis for each graph is the labor status with preterm nonlabor on the left, preterm labor in the middle, and term labor on the right. The clusters containing ST3, c-fos, and PGDH are shown.

 

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  		Table 3. Human Myometrial Genes that Are Up-Regulated at Least 2-Fold in Both Preterm and Term Labor Compared with Preterm Nonlabor

 

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  		Table 4. Human Myometrial Genes that Are Down-Regulated at Least 2-Fold in Both Preterm and Term Labor Compared with Preterm Nonlabor

 
Our analysis of mouse pregnancy centered upon days associated with significant changes in prostaglandin synthetic enzymes. In the human, changes in prostaglandin biosynthesis are also associated with the onset of labor. In contrast to our murine data, we saw no change in COX-1 or COX-2 mRNA in human myometrium. Instead, we found a significant reduction in prostaglandin dehydrogenase (PGDH) expression, an enzyme that catalyzes the conversion of prostaglandins to inactive metabolites (Fig. 6Go, Table 4Go). Other changes in human prostaglandin-related genes include a reduction in the prostaglandin E2 receptor, EP3a1, and phospholipase C-{epsilon} expression (Table 4Go). Although SOX4 did not significantly increase as it did in the mouse, there was a trend toward increased expression in the women who were in labor, both term and preterm. ST3 also shows an increase in preterm laboring human myometrium as was seen with the mouse data (Fig. 6Go). As further validation of our human chip data, our observation that c-Fos increases in expression in the laboring human myometrium has been previously suggested by other methods (Fig. 6Go) (28, 29).

In a manner analogous to our evaluation of the murine microarray data, we investigated expression patterns of GenMAPP functional groups of human genes by hierarchical analyses (Fig. 7Go). Again, approximately half of the groups displayed similar numbers of up-regulated and down-regulated genes. In marked contrast to the mouse data, the immune/inflammatory, protease/protease inhibitor, and cell adhesion groupings revealed a preponderance of genes whose relative pattern of expression increased rather than decreased (Fig. 7Go). Somewhat surprisingly, whereas only 77 of the 478 genes met the criteria of changing at least 2-fold and achieving statistical significance in both preterm and term labor, the hierarchical analyses reveal similar trends in the overall pattern of gene expression in the groups of preterm and term labor samples, although of somewhat differing magnitude (Fig. 7Go).



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  		Fig. 7. Functional Grouping of Hierarchical Clustered Human Genes from Gravid Myometrium

The genes shown in Fig. 6Go were divided into functional groups based on GenMAPP, and relative changes in their expression level were indicated by a color code. Red indicates that the gene is up-regulated, and green indicates that the gene is down-regulated. The brighter the color, the larger the change. All changes are compared with PTNL. Black indicates no change from PTNL. The first column is PTNL (all in black), the second column is preterm labor, and the third column is term labor.

 
To identify changes in gene expression most reliably associated with labor, we extracted those genes from the data set that showed at least a 2-fold up- or down-regulation in both preterm and term laboring myometrium compared with the preterm nonlabor specimens (Tables 3Go and 4Go). Forty-two genes were up-regulated, and 35 genes were down-regulated in both of the preterm and term laboring myometrium data sets.

We next examined whether there were any uterine genes similarly regulated in both humans and mice during pregnancy. Table 5Go shows the 12 genes that are similarly regulated in human term or preterm laboring myometrium and in murine gravid uterus samples.


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  		Table 5. Mouse and Human Uterine Genes that Are Similarly Regulated Just before Delivery

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The microarray studies described here were initiated to identify novel targets important in the process of parturition. Although many genes would be expected to change during the course of gestation, comparison of gene expression profiles among members of the same species as well as across species may identify an enriched population of genes truly important for defining the onset of labor. The genes whose patterns of expression were validated by in situ hybridization analysis strongly support the changes detected by microarray, and other genes not validated by in situ hybridization have been previously demonstrated to be induced or repressed in a manner consistent with the chip findings. Thus, we expect that the patterns of expression for the many novel genes that we have not evaluated are accurate, making this data set a valuable resource for many investigators interested in parturition biology.

Prostaglandins have been shown to be essential for normally timed labor in mice (5, 6, 30, 31). Using conventional protein and RNA blot approaches for evaluation of prostaglandin synthetic enzymes known to be essential for labor in mice, we have recently demonstrated no change in cytosolic phospholipase A2, induction of COX-1, and induction of prostaglandin F synthases (32). The mouse microarray data confirm an increase in COX-1 mRNA levels between gestational d 13.5 and 16.5 and no change in the expression of cytosolic phospholipase A2. The murine chips do not detect transcripts for prostaglandin F synthases. In functional clustering of the genes, COX-1 was the only gene involved in prostaglandin metabolism to significantly change in the gravid mouse uterus. This is consistent with a role for COX-1 as the key regulator of the synthesis of prostaglandins important during murine parturition. In humans, prostaglandins are known to play an important role in labor because administration of prostaglandins and prostaglandin synthesis inhibitors affect the progression of labor (33, 34, 35). Consistent with Western blot analyses (33), the human microarray did not show a change in COX-1 or COX-2 expression but did show several genes involved in prostaglandin metabolism that were down-regulated in the human laboring myometrium. PGDH, which inactivates prostaglandins, decreased in term laboring samples. In the preterm labor samples, the trend for PGDH expression was decreased, but this was not statistically significant. Although our mouse microarray did not show significant hybridization to PGDH, we have previously shown that PGDH protein decreases during mouse gestation (32). In addition, there was a significant decrease in human term laboring samples and preterm laboring samples of EP3a1, one of the receptors for prostaglandin E2.

Our study identified 504 genes that changed significantly between d 13.5 and 19.0 gravid mouse uterus; of these, 23 were transcription factors. Elf3, which was present only in the endometrium, was up-regulated at the same time as COX-1. Because COX-1 is also an endometrial protein, we further evaluated the possibility that Elf3 plays a role in regulating COX-1 expression. Indeed, we found putative binding sites in the COX-1 promoter and that cotransfection of an Elf3 expression vector with a COX-1 reporter plasmid significantly augmented COX-1 promoter activity. These studies demonstrate the power of the microarray approach, as Elf3 had not been previously implicated in the regulation of COX-1 gene expression or the parturition process. ELF3 was not present on the human microarray to determine whether similar changes occurred during human pregnancy.

Sox4 expression was also up-regulated in the gravid uterus, but in contrast to Elf3, it was located in the myometrium. Because Sox4 was up-regulated in the murine myometrium at term, it may play a role in the regulation of the contraction-associated proteins such as the oxytocin receptor, prostaglandin receptors, and connexin-43, which are known to be induced just before delivery. Analysis of the oxytocin receptor (GenBank accession no. D86331) and connexin 43 (GenBank accession no. U17892) promoter regions with Match 1.0 revealed potential SOX binding sites at positions 601–614 and 1119–1132 for the oxytocin receptor promoter (transcription initiation site at 31) and positions 878–891, 917–926, 1176–1189, 1318–1331, and 2172–2185 for the connexin 43 promoter (transcription initiation site at 1690). Inactivation of Sox4 is embryonically lethal due to the requirement of Sox4 for the normal development of the heart (20). Our studies suggest that conditional inactivation of Sox4 in the myometrium to determine its role in murine labor may be warranted. SOX4 expression on the human chip did not show a consistent change in expression level. However, the trend was toward increased expression in the myometrium of women during term and preterm labor.

In the human microarrays, the general pattern of expression of genes modulated in preterm labor and term labor were concordant; however, there were only 77 genes that achieved statistical significance and changed by at least 2-fold in both term and preterm labor samples. Of these genes, only six are known transcription factors: c-Fos, MafF, enigma, estrogen receptor factor-1, LIM protein, and Jun B. Both c-Fos and c-Jun have been shown to be expressed in uterine myometrial and endometrial cells (36, 37). c-Fos and c-Jun regulate gene transcription by heterodimerizing and binding to the AP-1 sites and have been implicated in the regulation of genes of the laboring myometrium such as connexin 43 (17, 38). Studies from myometrium isolated from women undergoing cesarean section have shown that c-Fos may be induced during human labor (28, 29). Our data confirm that c-Fos is up-regulated in women in labor, both term and preterm. Because the c-Fos null mouse demonstrates abnormal gametogenesis, the effects of c-Fos deficiency on parturition are unknown (21).

MafF is a transcription factor member of the musculoaponeurotic fibrosarcoma oncogene family that was up-regulated 2.7-fold in human term laboring specimens and 3.1-fold in preterm labor data sets (39, 40). Human MafF had previously been shown to be up-regulated in myometrium after term delivery but not in myometrium from early gestation compared with the nonpregnant myometrium. Although MafF itself does not contain a transcription activation domain, it may regulate transcription by dimerizing with other Maf proteins or b-Zip proteins. Human MafF binds to the promoter of the oxytocin receptor gene, and thus may contribute to normally timed labor (41). c-Maf is a related gene, present on the mouse chip, that increased 1.7-fold from gestational d 13.5–19.0 (data not shown).

Only four genes known to be involved in the cell cycle, p57Kip2, Cdc2a, Cdc20, and regulator of G protein signaling, displayed significant changes in expression in murine uterus during late pregnancy. p57Kip2 is a paternally imprinted gene that maps to human chromosome 11p15.5 and mouse chromosome 7. This region in humans has been linked to both sporadic cancers and Beckwith-Wiedemann syndrome (22, 42, 43, 44, 45). Female mice that are heterozygous for p57Kip2 have been shown to deliver their pups 2 d early (23). Mice that are homozygous knockouts of p57Kip2 and survived to weaning age had immature uteri. Although the human data for p57KIP2 was variable and not statistically significant, the average of the expression levels showed an increase in p57KIP2 levels in the women in term labor.

Although genes involved in tissue remodeling and cell adhesion are not likely targets for the initiation of the parturition cascade, some of these genes are likely to play an important role in parturition. ST3 expression has been associated with the significant tissue remodeling during embryonic implantation and embryonic development, though its substrate remains unknown (24, 25). ST3 expression has also been associated with the postpartum involution of the uterus. ST3 expression increased significantly in the mouse uterus throughout pregnancy. This is similar to the increased expression of ST3 seen in women in preterm labor.

In summary, we have described the pattern of uterine gene expression during late pregnancy in both mice and humans. We identified 12 genes that changed similarly in both gravid mouse uterus and in either preterm or term human laboring myometrium. All of these genes significantly changed in the human preterm labor samples, but only three changed significantly in the term labor samples. These data show that relatively few of the genes that significantly change are common between mice and humans at term. In part, this may reflect different contributions of genes expressed in myometrial and nonmyometrial uterine cell populations, as the human samples are enriched for the myometrium. Nonetheless, the significantly changing human genes should still be represented in the mouse data if regulated in a similar fashion. Although progesterone withdrawal is the key regulatory event needed to start active labor in mice, progesterone does not decrease before labor in humans. Several studies have argued that functional progesterone withdrawal could occur in the human myometrium due to changes in local metabolism or action (46, 47). If this were the case, a greater degree of conserved gene expression may be expected than we demonstrate in our studies. Indeed, the most highly induced gene we find during both human preterm and term labor is decidual protein induced by progesterone, a progesterone-induced protein, further arguing against functional progesterone withdrawal. The discordant regulation of inflammatory molecules and proteases comparing humans and mice suggest a much more prevalent contribution of inflammation than progesterone withdrawal to human labor. The similarity in the overall pattern of gene regulation between idiopathic preterm and term labor in the human samples further suggests that idiopathic preterm labor occurs by a mechanism similar to that precipitating labor at term. Finally, our studies identify several genes that could not have been anticipated to be essential for parturition that should prove important for ongoing physiological studies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animal Husbandry
All mouse protocols were in accordance with National Institutes of Health guidelines and approved by the Animal Care and Use Committee of Washington University School of Medicine (St. Louis, MO). All mice used were 8–16 wk old and of a C57BL/6 genetic background. Mice were housed on a 12-h light, 12-h dark cycle with ad libitum access to rodent chow and water. Matings of estrous females to stud males were confirmed via detection of a copulation plug, with the morning of plug detection designated as d 0.5 of gestation. Plugged females were removed from the male cage to ensure accurate gestation timing.

RNA Isolation
Nongravid, gestational d 13.5, 16.5, and 19.0 mice were killed by CO2 inhalation, and then the uteri were isolated. For gravid samples, the fetuses were removed, and half of the uterus was snap-frozen in liquid nitrogen and stored at -80 C until RNA was isolated. Half of the mouse uterus was fixed by immersion in 4% paraformaldehyde for 24 h, cryoperserved in 10% sucrose in PBS, and embedded in OCT compound (Sakura Finetek, Torrance, CA) for sectioning on a cryostat. A small section of uterine myometrium was harvested as described below from women undergoing cesarean delivery. The human samples were rinsed in phosphate-buffered sodium chloride solution and then snap-frozen in liquid nitrogen and stored at -80 C until RNA was isolated. RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA), after the manufacturer’s instructions. Integrity of the RNA was confirmed by running 2 µg on a 1.2% agarose gel.

Mouse
Uteri were harvested on gestational d 13.5, 16.5, and 19.0. The first set of data was collected in duplicate and pooled together. The second and third sets of data consisted of different individual mouse samples. A total of three chips were used per time point.

Human
The study was in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Washington University School of Medicine. Pregnant women between 26 and 41 wk of gestation were eligible to participate if they required a cesarean delivery, had no evidence of infection, and consented to participate in the study. Gestational age was determined by the last menstrual period and early ultrasonographic examination. Preterm samples (n = 3 nonlaboring; n = 3 laboring) were obtained from women who were between 26 and 33 wk of gestation. Term labor samples (n = 3) were obtained from women between 37 and 41 wk of gestation. Tissue samples were obtained immediately after delivery of the fetus. A myometrial sample (~1 cm3) was removed from the upper portion of the low-segment uterine incision and separated from any serosal or decidual components.

In Situ Hybridization
In situ hybridization using an {alpha}-33P-uridine triphosphate-labeled probe for the indicated gene was performed after a modification of the procedure of Simmons (48). The radiolabeled probes were prepared by incubating 2 µg of linearized template with the appropriate polymerase as previously described (49). Elf3 was linearized with EcoRI and T3 polymerase was used (Incyte Genomics, St. Louis, MO; GenBank accession no. AA832961). Sox4 was linearized with NotI and T7 polymerase was used (ATCC, Manassas, VA; GenBank accession no. AW988814). c-Fos was linearized with EcoRV and T7 polymerase was used (Incyte Genomics, GenBank AW208619). ST3 was linearized with EcoRI, and T3 polymerase was used (ATCC; GenBank accession no. AI120903). The XhoI/ApaI fragment of p57Kip2 (Incyte Genomics, GenBank accession no. AI874732) was ligated into the XhoI/ApaI sites of pBluescript II SK (Stratagene, La Jolla, CA). It was then linearized with KpnI and the T3 polymerase was used. Uterus sections were hybridized to the appropriate riboprobe in a humidified chamber for 20 h at 60–65 C. After washes and vacuum drying, slides were exposed to BioMax MR film (Eastman Kodak, Rochester, NY) for 4–10 d, emulsion-dipped and developed after an additional 3–6 wk for visualization of hybridizing areas. Slides were counterstained with hematoxylin and eosin.

Microarray Hybridization
Gene chip analysis was performed using the Affymetrix Mu11K SubA and SubB chips for murine samples and the HG-U95A chips for human samples. Probes for microarray analysis were prepared using 20 µg of total RNA. First strand cDNA synthesis was performed using a T7-(T)24 primer (Genosys, The Woodlands, TX) and Superscipt II reverse transcriptase (Invitrogen Corp., Carlsbad, CA). Second strand synthesis was performed using DNA polymerase I, Escherichia coli DNA ligase, and ribonuclease H (Invitrogen Corp.). Hybridization reactions were performed using 15 µg of biotinylated cRNA prepared from the entire cDNA reaction. After hybridization, the arrays were processed using a GeneChip Fluidics Station 400 (Affymetrix). Specifically bound probe was detected by incubating the arrays with streptavidin phycoerthryin (Molecular Probes, Eugene, OR) and scanning the chips using a GeneArray Scanner (Agilent, Palo Alto, CA). For further details see Novak et al. (50).

Microarray Analysis and Cluster Analysis
The scanned images were analyzed using the GeneChip Analysis Suite 4.0 (Affymetrix). These values were processed by ArrayStat (Imaging Research Inc., Ontario, Canada) using a proportional model for random error with mean rescaling (50). Outliers were removed by examining the kurtosis of the distribution of signal intensities measured for each from each GeneChip and represented approximately 3% of the data values. The deviation of each data set from a normal distribution was assessed by using quantile-quantile plots. Statistical significance was tested by t test using the human PTNL (preterm nonlabor) or murine d 13.5 gestation data sets as a reference. False positive error correction was performed to maintain a 5% false discovery rate (51). The genes were then filtered to exclude those that changed less than 2.0-fold in at least one comparison. A total of 504 genes for mouse and 478 genes for human were statistically significant from our analysis.

A heuristic method of clustering analysis was developed to group similarly regulated genes in time series or for samples with multiple discrete experimental conditions (52). Experimental measurements were performed at three different time points for the murine data (d 13.5, 16.5, and 19.0) and for three conditions (PTNL, preterm labor, and term labor) for the human samples. Fold changes were obtained by using the d 13.5 or PTNL measurement as a control value for each gene. Clustering was then performed as follows: 1) the genes were separated into two categories, up- or down-regulated, determined by the slope between the first two data points. 2) Genes in each group were divided into four clusters by comparing the sign and value of the slope measured between the first and second data points as well as the second and the third data points. Figures 1Go and 6Go show the resultant clusters for the mouse and human data, respectively. The different cluster groups show distinct gene regulation patterns and distinct changes in their expression levels. Analysis was carried out using MATLAB 6.12 (MathWorks Inc., Natick, MA). Functional categories were assigned for each group member using GenMAPP (53) and by searching online databases (Unigene, Online Mendelian Inheritance in Man (OMIM), Locuslink, and PubMed). Using these methods, functional categories were assigned for 177 unique murine genes and 248 unique human genes (Figs. 2Go and 7Go).

Transfection Analysis
The COX-1 promoter region extending from -2.4 kb upstream of the transcription initiation site through bp 193 of exon 2 was generated by PCR of mouse genomic DNA and cloned into a nuclear ß-galactosidase expression vector, pnlacF (54, 55), to generate p5'COX1-E2. Accuracy of the amplified sequences was confirmed by sequence analysis. To ensure that the ß-galactosidase sequences were read in frame, the endogenous mouse COX-1 initiation codon was mutated to TAG. For transfection experiments, Chinese hamster ovary cells were seeded in six-well plates at a density of 2 x 105 cell/well in Ham’s F-12 medium with 10% fetal bovine serum for 24 h. Chinese hamster ovary cells were transfected with a GenePORTER/DNA mixture containing 10 µl of GenePORTER (Gene Therapy Systems, Inc., San Diego, CA), 2 µg p5'COX1-E2 or pnlacF, 5 ng of pGL3 (luciferase control plasmid; Promega, Madison, WI), and pCMV-mELF3 (27) in a total volume of 1 ml of Ham’s F-12 without serum. After a 4-h incubation, 1 ml of media with 20% fetal bovine serum was added. After 48 h, cells were collected and assayed for luciferase and ß-galactosidase activity. ß-Galactosidase activity normalized to relative luciferase activity was determined for triplicate samples with data shown as mean ± SEM. The results are representative of two independent experiments.


   ACKNOWLEDGMENTS
 
We thank Genome Quebec for support of the DNA chip facility at the Montreal Genome Centre, Sherri Vogt (Washington University) for technical assistance, Catherine Côté (Montreal Genome Centre) for assistance with GenMAPP library files, and Dr. Angie Rizzino (University of Nebraska Medical Center) for the Elf3 expression vector.


   FOOTNOTES
 
This work was supported by grants from the March of Dimes (to L.J.M.), Rockefeller Brothers Fund (to L.J.M.), and National Institutes of Health (to K.E.B.), and the Lawson Wilkins Pediatric Endocrine Society Abbott Clinical Scholars Award (to K.E.B.). R.S. is a recipient of a fellowship from the Canadian Institute of Health Research. T.J.H. is a recipient of an Investigator Award for the Canadian Institute of Health and the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.

K.E.B. and Y.N. contributed equally to this work.

Abbreviations: COX, Cyclooxygenase; PGDH, prostaglandin dehydrogenase; PTNL, preterm nonlabor; ST3, stromelysin-3.

Received for publication January 8, 2003. Accepted for publication May 19, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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