Molecular Human Reproduction, Vol. 9, No. 11, 681-700,
November 2003
© 2003 European Society of Human Reproduction and Embryology
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Human myometrial adaptation to pregnancy: cDNA microarray gene expression profiling of myometrium from non-pregnant and pregnant women
Submitted on May 5, 2003; resubmitted on June 10, 2003. accepted on June 20, 2003
Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9032, USA
1 To whom correspondence should be addressed. e-mail: william.rainey{at}utsouthwestern.edu
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The human uterus undergoes profound physiological tissue remodelling during pregnancy. In the myometrium, altered gene expression must underlie these extensive molecular and structural changes. The purpose of this study was to compare expression profiles of pregnant and non-pregnant myometrium, in order to identify genes that participate in this process. mRNA from 14 non-pregnant and four pregnant human myometrial samples were analysed using a human UniGEM V microarray with 7075 cDNA elements. A total of 602 transcripts from the microarray were up-regulated
2.0-fold in pregnant myometrium, with 37 transcripts up-regulated 4.0-fold. In contrast, eight transcripts were down-regulated 2.0-fold in pregnancy. To ensure accurate representation of differential gene expression, Northern blot analyses using total RNA from 16 samples of non-pregnant and pregnant myometrium were used to examine mRNA levels for four of the genes that were differentially expressed by microarray analysis, namely plasminogen activator inhibitor type 1 (PAI-1), milk fat globule-EGF factor 8 protein (MFGE8), secreted frizzled-related protein 4 (sFRP4) and estrogen receptor 
(ER). On the microarray these transcripts were up-regulated 7.5-fold for PAI-1 and 4.9-fold for MFGE8 in pregnant myometrium, and down-regulated 3.7-fold for sFRP4 and 2.9-fold for ER in pregnancy. Northern blot analyses confirmed these changes. Our findings suggest that microarray technology is a useful tool for examining global changes in gene expression that occur as the myometrium differentiates from non-pregnant to pregnant status. Defining these changes provides new insight into the structural and functional adaptations of human myometrium during pregnancy. Key words: gene expression/microarray/myometrium/non-pregnant/pregnant
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Introduction |
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The anatomical, physiological and biochemical adaptations that take place in the uterus during the short span of human pregnancy represent one of the most active processes of tissue remodelling in normal adult physiology. The uterus can achieve 500- to 1000-fold greater capacity during pregnancy, and its weight increases from 50 to 1100 g (Ramsey, 1994). The myometrium is the main component in the enlargement of the uterus during pregnancy. The myometrium is the distinct muscular layer of the uterine wall, which is involved in contraction during labour. The myometrium consists predominantly of smooth muscle cells but also contains fibroblasts, blood and lymphatic vessels, immune cells and connective tissue. The connective tissue, or stroma, provides a supportive matrix for the bundles of smooth muscle, and a framework that expands as the uterus distends during gestation. Although uterine growth during the first few weeks of pregnancy is accomplished by increased numbers of smooth muscle cells (i.e. hyperplasia) and a smaller contribution from increased cell size (i.e. hypertrophy), the predominant growth of the uterus during pregnancy is by way of stretch-induced myometrial hypertrophy (Ramsey, 1994). This ongoing process of stretch-induced tissue remodelling and smooth muscle hypertrophy is accompanied by the lack of uterine contractions during most of gestation to accommodate the developing fetus (phase 0 of parturition). Phase 1 of parturition represents myometrial activation (Challis, 2001). The final stages of pregnancy are characterized by increases in spontaneous low-amplitude contractions that gradually increase in frequency, rhythmicity and strength, normally culminating in labour and delivery of the fetus at term (phase 2 of parturition).
Utilizing animal models and tissues from pregnant women at term, several gene products have been identified as markers of the transition from uterine quiescence to myometrial activation. These markers, most of which have been identified in studies of labouring myometrium, include increased expression of myometrial oxytocin receptors (Soloff et al., 1979; Helmer et al., 1995; Fuchs et al., 1998), cyclooxygenase-2 (Zuo et al., 1994; Slater et al., 1999), interleukin-8 (Osmers et al., 1995; Elliott et al., 2000), and prostaglandin receptors (Brodt-Eppley and Myatt, 1999), as well as increased expression of myometrial gap junction components (Garfield et al., 1988; Lefebvre et al., 1995; Kilarski et al., 1998). In addition, previous functional genomic studies of myometrium have provided comparisons between human pregnant myometrium before and after the onset of labour using cDNA macroarrays (Aguan et al., 2000), and non-pregnant mouse myometrium compared with models of preterm labour using cDNA microarrays (Muhle et al., 2001). Nevertheless, very few investigations have identified major changes in gene expression during adaptations of the myometrium to pregnancy. For example, the expression of smooth muscle myosin heavy chain, smooth muscle actin, calmodulin, myosin light chain kinase (MLCK) and myosin phosphatase are similar in non-pregnant and pregnant women (Word et al., 1993). Activity of type II phosphatase is down-regulated in pregnancy, and smooth muscle caldesmon is increased in pregnancy (Word et al., 1993). Gap junctions are few in number during most of pregnancy (Garfield et al., 1988; Chow and Lye, 1994). Lastly, extracellular matrix components including collagen types I and III, and fibronectin, are up-regulated in pregnancy (Stewart et al., 1995). It is likely that these genes represent only a fraction of the changes in gene expression in the pregnant myometrium.
Standard techniques used to study individual proteins or transcripts, namely Western analysis, Northern analysis and semi-quantitative and real-time PCR, do not allow a general assessment of gene expression. To better understand global gene expression alterations underlying the extensive changes occurring in myometrial adaptation to pregnancy, we herein compare the gene expression profiles between non-pregnant and pregnant myometrial tissue using high density cDNA microarray technology. In contrast to studies that have focused solely on myometrial changes after the onset of labour, we believe that more detailed knowledge of the changes in human myometrial function during pregnancy will further our understanding of the maintenance of pregnancy, and the initiation of both term and preterm labour. The present study thus represents the first investigation of global gene expression differences between non-pregnant and pregnant human myometrium.
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Non-pregnant and pregnant myometrial tissues
Fourteen samples of non-pregnant myometrium for microarray analysis were obtained from uteri of women undergoing abdominal or vaginal hysterectomy for benign gynaecological indications, and seven non-pregnant myometrial specimens were also obtained in a similar manner and used for Northern analyses (Table I). A single patient was found to have a malignancy (leiomyosarcoma) on postoperative pathology, but this had not infiltrated normal myometrium remote from the tumour site. Four pregnant myometrial specimens for microarray analysis were obtained from the superior margin of the lower uterine segment incision at the time of Caesarean section performed prior to the onset of labour. For Northern analyses, nine samples of pregnant myometrium were obtained from either the fundus or the lower uterine segment of uterine specimens obtained by Caesarean hysterectomy performed prior to the onset of labour (Table II). All pregnancies had progressed to term, between 39 and 40 weeks of gestation. All tissues were obtained in accordance with the Donors Anatomical Gift Act of the State of Texas after written informed consent was obtained, and all protocols were approved by the Institutional Review Board (IRB) of the University of Texas Southwestern Medical Center at Dallas. All myometrial tissues were obtained from grossly and microscopically normal areas remote from the endometrium, cervix or leiomyomas. Tissues were dissected free of serosa, connective tissue and major blood vessels, rinsed three times in phosphate-buffered saline, and snap-frozen in liquid N2 before use. Although the non-pregnant and pregnant myometrial samples were carefully dissected by experienced investigators to minimize decidual inclusion, we cannot discount the possibility of residual decidual tissue as a contaminant in comparisons of myometrial gene expression. Further, populations of trophoblastic cells exist within the myometrium, including deep invasion into the myometrial arterial vasculature.
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RNA extraction
Myometrial tissues were pulverized in liquid nitrogen and then homogenized in guanidinium isothiocyanate, followed by the isolation of total RNA (Chirgwin et al., 1979). Purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis prior to use. For microarray analysis, total RNA was used to isolate poly(A)+ RNA using two successive oligo (dT) cellulose columns, following the manufacturer’s directions (Amersham Biosciences, USA).
cDNA microarray
cDNA probe synthesis, hybridization with the UniGEM V microarray Version 1.0 and signal analysis were conducted by Incyte Genomics (St Louis, MO, USA) according to the manufacturer’s protocols (Yue et al., 2001; Reynolds, 2002). The cDNA microarray for Version 1 consisted of 7075 unique probes arrayed onto glass slides. Additionally, 192 internal control probes were included on the microarray to assess the quality of probe generation and hybridization. Isolated non-pregnant and pregnant myometrium poly(A)+ mRNA (200 ng) was reverse-transcribed and labelled with the fluorescent dyes Cy3 for non-pregnant myometrium, or Cy5 for pregnant myometrium (Peron Technologies, USA). The two labelled cDNA samples for non-pregnant and pregnant myometrium were combined and purified, and were simultaneously applied to the array. Following incubation, the microarray was rinsed and laser-scanned using 532 nm for Cy3 and then at 635 nm for Cy5. A signal intensity of 2.5-fold over background was used for data analysis. Data were normalized for difference in signal between the Cy3 and Cy5 channels to compensate for any differences in labelling and detection efficiency which might bias observed expression ratios. The ratio of the two fluorescent intensities was then used as a quantitative measurement of the relative gene expression between the two tissue samples.
Northern blot analysis
Samples of RNA (10 µg) were separated by electrophoresis on 1% agarose gels in the presence of formaldehyde. RNA was transferred to a nylon membrane (Amersham Biosciences) by overnight blotting at 10 V and was cross-linked under UV light. Hybridizations were performed at 42°C for 16 h, using cDNA probes. A 381 bp fragment of the plasminogen activator inhibitor type 1 (PAI-1) gene (GenBank accession no. M16006, nucleotides 531–912), a 549 bp DNA fragment of the milk fat globule-EGF factor 8 protein (MFGE8) gene (GenBank accession no. NM_005928, nucleotides 300–849), a 666 bp fragment of the human secreted frizzled-related protein 4 (sFRP4) gene (GenBank accession no. AF026692, nucleotides 252–918) and a 599 bp fragment of the estrogen receptor (ER) gene (GenBank accession no. X03635, nucleotides 5001–5600) were cloned in pCRII Topo vector (Invitrogen, USA) and used to generate labelled probes. RNA hybridization with a cDNA probe against 18S ribosomal RNA (18S rRNA), generated using a 1212 bp cDNA template (Ambion, USA), was used to ensure RNA integrity and equal loading of lanes (data not shown). After hybridization, the radioactivity on the transfer membrane was counted using the AMBIS Radioanalytic Imaging System (AMBIS Systems, USA). All results were normalized to 18S rRNA levels prior to further analysis to allow standardized comparison of mRNA abundance for each of the transcripts studied.
Statistical analysis
Quantitative radiometric Northern analysis data generated by the AMBIS Imaging System were analysed using the Mann–Whitney U-test, comparing counts per minute for non-pregnant and pregnant myometrial RNA specimens. A two-tailed significance level of 0.05 was used.
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Results |
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RNA isolated from a total of 14 samples of non-pregnant myometrium and four samples of pregnant myometrium was analysed using a genomic expression microarray (GEM). The complete gene expression data set is available in the Gene Expression Omnibus public data repository, located at the Internet site http://www.ncbi.nlm.nih.gov/geo/, with accession number GSM6791 (NCBI, Bethesda, MD; Edgar et al., 2002). In all, 97% of the genes examined were expressed at a level
2.5-fold that of background signal. A plot of all the gene comparisons from the array analysis is displayed in Figure 1. Transcripts differentially expressed 4.0-fold higher in pregnant myometrium and 2.0-fold higher in non-pregnant (i.e. 2-fold lower in pregnant) myometrium are represented by red dots. The vast majority of genes tested were expressed to a similar degree in non-pregnant and pregnant myometrium. From the GEM microarray analysis, 602 transcripts (8.5% of the probes in the microarray) were increased 2.0-fold in pregnant myometrium, with 164 transcripts (2.3%) increased 2.5-fold. Thirty-seven transcripts (0.5%) were differentially expressed 4.0-fold in pregnant myometrium (Table III). We selected a 4.0-fold threshold for up-regulation to focus our discussion on the most highly up-regulated genes in pregnant myometrium; however, the full list of genes up-regulated 2.0-fold is shown in the Appendix. The single most differentially expressed transcript was insulin-like growth factor binding protein 1 (IGFBP-1), which was increased 20.5-fold in pregnant myometrium. Other genes whose expression was significantly up-regulated in pregnant myometrium included insulin-like growth factor 2 (4.1-fold) and wingless-type MMTV integration site family member 5A (WNT5A; 4.5-fold). In comparison, only eight transcripts (0.1%) were significantly down-regulated (decreased 2.0 fold) in pregnant myometrium (Table IV). sFRP1 and sFRP4 were decreased in pregnant myometrium, 4.4- and 3.7-fold, respectively. As previously reported, ER was decreased 2.9-fold in pregnant myometrium.
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To confirm that the microarray data accurately represented differentially expressed genes in human myometrial tissues during pregnancy, we compared mRNA expression of selected genes in multiple individual samples of myometrium from non-pregnant and pregnant women using traditional Northern analysis. Two transcripts with significantly increased expression in pregnant myometrium, plasminogen activator inhibitor type 1 (PAI-1) and milk fat globule-EGF factor 8 protein (MFGE8) were further studied by Northern analysis using RNA isolated from six non-pregnant and six pregnant myometrial tissues for PAI-1, and from five tissues per group for MFGE8. The laser fluorescence scan for Cy3 (non-pregnant myometrium) and Cy5 (pregnant myometrium) showed substantially increased expression of PAI-1 (7.5-fold) and MFGE8 (4.9-fold) in pregnant myometrium (Figure 2). Northern analysis was consistent with the microarray data. PAI-1 mRNA levels, expressed relative to 18S rRNA, were increased 5.2 ± 1.5-fold in pregnant myometrium (mean ± SEM; P = 0.02). MFGE8 mRNA was increased 3.8 ± 0.9-fold (P = 0.01) in pregnant myometrium (Figure 2).
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Two transcripts from the microarray with decreased expression in pregnant myometrium were also selected for further analysis, secreted frizzled-related protein 4 (sFRP4) and ER
. Their respective fluorescence scans demonstrate 3.7- and 2.9-fold decreased expression in pregnant myometrium (Figure 2). Northern analysis using RNA obtained from five non-pregnant and five pregnant myometrial tissues showed that sFRP4 expression was variable in non-pregnant women, and was higher in two proliferative phase myometrial specimens than three from the secretory phase (data not shown). In pregnant women, sFRP4 mRNA levels were consistently down-regulated, resulting in differential sFRP4 mRNA expression that was decreased 15.3 ± 7.1-fold (P = 0.01) in pregnant myometrium. Northern analysis using six tissues per group showed that ER mRNA was decreased 3.8 ± 0.8-fold (P = 0.02) in pregnant myometrium (Figure 2).
An alternative approach to analyse the microarray expression data is to examine the transcripts with the highest signal intensities in both pregnant and non-pregnant myometrium, representing a semi-quantitative measure of mRNA abundance. Table V shows the 10 transcripts with the highest signal intensity for pregnant myometrium, and Table VI those for non-pregnant myometrium. Eight genes were highly expressed in both tissues, notably 2 actin and transgelin. Two genes, tropomyosin 2 and caldesmon 1, had high signal intensities only in pregnant myometrium, and were also strongly differentially expressed, 3.4- and 4.7-fold higher respectively, in pregnant myometrium. Another two genes, iron-responsive element binding protein 2 and an uncharacterized transcript (expressed sequence tag or EST), had high signal intensities only in non-pregnant myometrium, but these did not show significant down-regulation in pregnant myometrium.
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Discussion |
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High density cDNA microarray technology was used to analyse differences in gene expression between non-pregnant and pregnant myometrium. We have thus identified 45 transcripts differentially expressed either
4.0-fold higher in pregnant myometrium, or down-regulated 2.0-fold in pregnancy. These transcripts represent the major differences in myometrial gene expression between the two states. The vast majority of these genes have not been previously identified as being differentially expressed between non-pregnant and pregnant myometrium. Furthermore, we have discovered several uncharacterized transcripts (EST) that had high expression signals in myometrial smooth muscle. Even though some of these EST did not meet our specifications for differential expression between non-pregnant and pregnant myometrium, they may represent novel genes that have preferential expression in myometrial smooth muscle over other tissues. We believe that both known genes and EST represent potential determinants of the structural and functional phenotypes of non-pregnant and pregnant myometrium. Molecular and cellular studies of myometrium have provided some clues as to the most probable roles of the differentially expressed genes. Differentially expressed genes involved in growth and differentiation, signal transduction, cell adhesion, molecular recognition and cellular immunity, and structural and contractile genes are discussed below. We will also discuss the genes with the highest signal intensities, indicating the highest relative expression levels in either pregnant or non-pregnant myometrium.
Genes involved in growth and differentiation
Insulin-like growth factor signalling and prolactin
Genes of the insulin-like growth factor (IGF) signalling system, including the IGF, their binding proteins (IGFBP) and their related proteins (IGFBP-rP) are important regulators of growth and cellular proliferation. Further, IGFBP not only modulate IGF actions, but may act as regulators of cell growth independently of the IGF (Ferry et al., 1999). Members of this family with increased expression in pregnant myometrium by the present microarray analysis include IGF-2, which promotes DNA synthesis, cell proliferation and differentiation as well as having acute anabolic effects on protein and carbohydrate metabolism, and IGFBP-1 and IGFBP-3. IGFBP-1 may stimulate or inhibit IGF actions (Jones and Clemmons, 1995), whereas IGFBP-3 generally has inhibitory effects on IGF-regulated mitogenesis (Blat et al., 1989; Oh et al., 1993). The apparent contradictory increase in both a growth factor and an inhibitor of growth may be modulated by post-translational regulation of IGFBP-3 in myometrial cells (Huynh, 2000). IGF-1 was also differentially expressed 2.2-fold higher in pregnant myometrium. Although IGF-2 is expressed by the human placenta, after the first trimester its expression is limited to chorionic mesoderm and extravillous cytotrophoblasts, not the much more abundant syncytiotrophoblast (Han and Carter, 2000), reducing the likelihood that the observed differences simply reflect trophoblastic invasion. Further, the in-vivo importance of IGF-1 for myometrial growth has been clearly demonstrated in IGF-1 null mice, which have severe uterine hypoplasia (Baker et al., 1996). Thus increased expression during pregnancy of both IGF may act in regulating the growth of the fetus, as well as in effecting uterine growth to accommodate fetoplacental growth. In addition to members of the IGF family, increased expression of prolactin was found in pregnant myometrium. Although the decidua produces large amounts of prolactin (Riddick et al., 1978), endogenous prolactin may act as an autocrine growth factor in cultured human myometrial cells (Nowak et al., 1999). Prolactin has also been shown to have an increased level of expression in pregnant myometrium in the baboon (Frasor et al., 1999), suggesting that it may have a role in myometrial growth during pregnancy.
Wnt signalling pathways
Wnt proteins form a family of highly conserved secreted signalling molecules that regulate cell-to-cell interactions during embryogenesis. Their roles in differentiation include an important role in skeletal myogenesis (Cossu and Borello, 1999). Wnt proteins are ligands for the frizzled family of receptors (Bhanot et al., 1996). WNT5A was strongly differentially expressed in pregnant myometrium relative to non-pregnant myometrium, indicating a role for the Wnt pathway in adaptations of the uterus in pregnancy. However, this gene has not previously been examined in myometrium. Wnt signalling in animal models may lead to increased intracellular calcium via activation of phospholipase Cß. In pregnant myometrium, Wnt signalling could thus prepare for increased contractility. sFRP represent a family of secreted proteins that function as antagonists of Wnt for frizzled receptors (Finch et al., 1997; Dann et al., 2001). Transcript levels for the two sFRP family members present on the GEM array, sFRP1 and sFRP4 (formerly frpHE), were both significantly down-regulated in pregnant myometrium. Very few studies have examined the expression and possible role of sFRP family members in the myometrium. sFRP1 is expressed at low levels in non-pregnant human myometrium, and over-expressed in leiomyomata, particularly in response to estrogen stimulation (Fukuhara et al., 2002). In the mouse uterus, sFRP2 is expressed in endometrial stromal cells, but not in myometrium (Das et al., 2000). Thus other tissue systems studied may provide further insight into the potential role of these proteins in the uterus. First, in skeletal myoblasts activation of the Wnt pathway appears critical for embryonic differentiation (Cossu and Borello, 1999), suggesting that the drop in sFRP in pregnant myometrium may help maintain smooth muscle cell differentiation despite hyperplastic and hypertrophic growth in pregnancy. Second, sFRP have been shown to facilitate apoptosis through inhibition of the Wnt/Frizzled signalling network (Jones et al., 2000; Ko et al., 2002), and sFRP transcripts have been shown to induce apoptosis and become down-regulated in both human mammary and cervical carcinomas (Zhou et al., 1998; Ko et al., 2002). Thus, decreased expression of both sFRP1 and sFRP4 in pregnant myometrium may permit uterine growth during pregnancy by decreasing apoptotic myometrial cell death, and possibly also influences maintenance of contractile protein gene expression during pregnancy through Wnt signalling pathways. In summary, increased expression of WNT5A together with decreased expression of secreted Wnt signalling inhibitors indicates that the Wnt signalling network may be very important in myometrial adaptation to pregnancy.
Of note, variation between Northern analysis (15.6-fold) and microarray (3.7-fold) results was observed for sFRP4 differential expression. This may have been due to cross-reactivity of the probe used on the microarray with other sFRP genes with lower differential expression. We have previously observed that cDNA microarrays may not discriminate completely between gene family members with a high degree of sequence identity (Rainey et al., 2001). In addition, significant variability in mRNA levels in the non-pregnant tissues used for Northern analysis may be due to differences in menstrual cycle phase, with higher mRNA levels observed in the proliferative compared with the secretory phase tissues. A further source of variability may be the presence or absence of gynaecological pathology, such as stretch of normal myometrium in uteri enlarged by leiomyomas.
Plasminogen activator inhibitors
PAI regulate the functional activity of plasminogen activators, which are proteolytic enzymes that function during trophoblast implantation, vascular remodelling and maintenance of intervillous blood flow. PAI-1 has been demonstrated both in invading trophoblasts (Feinberg et al., 1989) as well as in pregnant myometrium (Uszynski et al., 2001). Thus, increased expression of PAI-1 in pregnant myometrium may represent trophoblastic invasion of myometrium. However, PAI-1 has also been demonstrated in smooth muscle and vascular endothelial cells of uterine tissue, with increased expression of PAI-1 mRNA in leiomyomas compared with adjacent myometrium (Sourla et al., 1996). Therefore, increased expression of PAI-1 in pregnant myometrium may be associated with hypertrophic and hyperplastic changes in pregnant myometrium, similar to the growth of smooth muscle in leiomyoma.
Milk fat globule-EGF factor 8
MFGE8 encodes a soluble integrin-binding protein with both epidermal growth factor (EGF)-like domains and coagulation factor VIII-like domains that mediate cell-to-cell interaction (Stubbs et al., 1990; Larocca et al., 1991). The human gene has 68% homology to mouse MFGE8 (Collins et al., 1997). In the mouse, it has been found to be secreted by a stromal cell type in early gonadogenesis (Kanai et al., 2000), whereas in humans it is diversely expressed in breast milk, some breast carcinoma, and some haematopoietic cells (Taylor et al., 1997; Kruger et al., 2000). In addition, MFGE8 is a major secretory product whose production is down-regulated by transfected cells over-expressing the gap junction protein connexin43, and in vitro this reduction in MFGE8 production decreased cell growth (Goldberg et al., 2000). However, connexin43 expression was increased in pregnant myometrium by microarray analysis (2.7-fold greater expression), as would be expected from the increase in gap junctions in pregnant myometrium even prior to the onset of labour; thus, this in-vitro relationship may not be a causal one. In summary, MFGE8 is currently considered an extracellular growth factor. The exact role of MFGE8 in the myometrium requires further research; however, we speculate that it may be one of the extracellular growth factors that facilitate expansion of the connective tissue framework supporting the hypertrophied bundles of smooth muscle in pregnancy.
Genes involved in signal transduction
The two estrogen receptor isoforms, and ß, exhibit distinct expression profiles in the non-pregnant and pregnant myometrium. ERß has been shown to increase during pregnancy while ER mRNA and protein levels fall as myometrium adapts to the pregnant state (Benassayag et al., 1999; Wu et al., 2000). The present study confirmed down-regulation of ER in pregnancy; however, the cDNA probe for ERß unfortunately did not meet the quality control parameters we established for microarray data. Interestingly, high affinity binding sites have only been reported in non-pregnant myometrium, suggesting that the switch from ER to ERß expression may result in the decreased myometrial responsiveness to circulating estrogen seen during pregnancy. Observations in knockout mice, where lack of ER alone (but not lack of ERß alone) disrupted both post-pubertal uterine development and uterine growth and genomic responses to estrogen (reviewed by Hewitt and Korach, 2003), also indicate the importance of ER rather than ERß in growth of the mouse uterus. Since the growth of the myometrium in the presence of increased estrogen during pregnancy is mainly due to hypertrophy, the proportion of ER and ERß may help to balance hypertrophy versus proliferation (Benassayag et al., 1999). Additionally, expression of the CX43 (GJA1) gene for the major gap junction protein, connexin43, has been found to be inhibited by estrogen in cultured human myometrial cells expressing mainly ERß, via AP-1 promoter sites (Wu et al., 2000). It has thus been suggested that myometrial ERß may block the induction of CX43 and other labour-associated genes during pregnancy, and that labour follows relative loss of ERß activity. Although we observed increased connexin43 expression in pregnant myometrium in the microarray analysis (2.7-fold higher), this represents a much lower level than is seen in labouring myometrium. In conclusion, nuclear hormone receptors such as ER that had differential expression by microarray analysis may play a role in regulating myometrial growth during pregnancy, and the dramatic switch from ER to ERß expression in the myometrium during pregnancy may help to delay labour until term.
Genes involved in cell adhesion, molecular recognition and immunity
A number of genes involved in cell adhesion and molecular recognition, and cellular immunity genes were shown to increase in expression during pregnancy using the microarray. These included the adhesion molecules tenascin C, chondroitin sulphate proteoglycan 2, and integrin, ß-like 1. Differentially expressed immune system gene transcripts included CD16 (the type II Fc 
receptor, associated with T cells) as well as the macrophage-associated receptor CD163, and the cytochrome b-245 ß gene, which is essential for normal neutrophil phagocytic activity (Teahan et al., 1987). Some of these genes may have interrelated roles. Cell adhesion molecules that may control leukocyte trafficking in human myometrium have been found to increase in expression during pregnancy, including intercellular adhesion molecule 1 (ICAM-1) and platelet endothelial cell adhesion molecule (PECAM) (Thomson et al., 2000). Recruitment of cytokine-producing immune cells may also be important for modulation of T-helper 1 versus T-helper 2 cellular immune responses, which may alter during pregnancy in response to local hormone and cytokine production, thus contributing to the immunological tolerance of pregnancy (Piccinni et al., 2000). In contrast to the genes up-regulated in pregnancy, down-regulation of carboxypeptidase A3, a molecular marker for mast cells, in pregnant myometrium may reflect the sharp decrease in the myometrial mast cell population in pregnancy (Thomson et al., 1999). However, the significance of this change is unknown.
Structural and contractile genes
The increased expression during pregnancy of the large number of localized and structural protein genes, including caldesmon and myosin, may indicate genes that play a role in producing and regulating contractions in pregnancy and labour. Caldesmon is a smooth muscle thin-filament-associated protein which inhibits generation of force by myosin (Marston and Redwood, 1991; Sobue and Sellers, 1991), although phosphorylated caldesmon may lose its ability to bind to actin and hence its inhibitory action after the onset of labour (Patchell et al., 2002; Li et al., 2003). Caldesmon mRNA was increased 4.7-fold in pregnant myometrium by microarray analysis, consistent with our previous report of a 4-fold increase of caldesmon protein in pregnant myometrium by immunoblotting (Word et al., 1993). Myosin genes that were differentially expressed higher in pregnant myometrium by microarray analysis included myosin heavy polypeptide 11 (3.1-fold), regulatory light polypeptide 5 (3-fold) and myosin IE (2.5-fold). Increased expression of smooth muscle myosin mRNA may be required to increase myosin expression proportionate to increased cell size, as we have previously shown that although the myosin content per myocyte or relative to DNA increases in pregnancy, the amount of myosin per milligram of protein is relatively unaltered compared with non-pregnant myometrium (Word et al., 1993). Thus a disproportionate increase in caldesmon expression relative to myosin during pregnancy may reflect net inhibition of contractile force maintenance by caldesmon prior to the onset of labour. This maintenance of uterine quiescence by increased caldesmon in pregnancy may be mediated by a concomitant decrease in myometrial calcium sensitivity (Li et al., 2003).
Genes with highest signal intensities
The data showing the transcripts with the highest signal intensities (Tables V and VI) add further information to the analysis of global gene expression. As might be expected, the highest expressed transcripts in pregnant myometrium included those coding for proteins involved in the smooth muscle contractile apparatus, including 2 actin, transgelin (also known as SM22; an actin-binding protein of uncertain function), tropomyosin 2, and caldesmon, which has already been discussed. Cysteine and glycine-rich protein 1 (CSRP1), which was highly expressed in both pregnant and non-pregnant myometrium, is a member of the CSRP family of genes encoding a group of LIM domain proteins. CSRP1 is expressed in both visceral and vascular smooth muscle in the mouse (Lilly et al., 2001), and has recently been shown to be involved in smooth muscle differentiation (Chang et al., 2003). Four uncharacterized sequences (EST) were also identified as highly expressed in pregnant myometrium.
Of note, an Incyte proprietary EST, designated by PD number 3097582, had the highest signal intensity in non-pregnant myometrium as well as the third-highest intensity of the genes expressed in pregnant myometrium. A partial sequence of this cDNA clone was obtained from Incyte, and was matched using the BLAST algorithm (Altschul et al., 1990) to locus 3q.28 on chromosome 3, consistent with UniGene cluster Hs.86945 (National Center for Biotechnology Information, Bethesda, MD, USA; http://www.ncbi.nlm.nih.gov/). This is 
10 kilobases 5' to the gene for IGF-2 mRNA-binding protein 2 (IMP-2; accession no. NM_006548), and may represent a novel gene. The second-highest expressed transcript in non-pregnant myometrium was iron-responsive element binding protein 2 (IREB2), an RNA-binding protein involved in the regulation of iron metabolism (Hentze and Kuhn, 1996). The role of IREB2 in non-pregnant myometrium has not been studied. The group of transcripts with the highest signal intensities in non-pregnant myometrium also included actin, transgelin, CSRP1 and five novel sequences or EST.
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In conclusion, use of high-density cDNA microarray technology to compare gene expression profiles of pregnant and non-pregnant myometrium has provided numerous new targets of study. The functions of a few of the genes from this microarray analysis have been identified in myometrium or other smooth muscle-containing tissues, but the majority of the genes showing differential expression have never been studied in the context of myometrial tissue. We have even identified some novel genes (EST) with preferential expression in myometrium. The majority of the change in gene expression occurred in pregnant myometrium, and further studies of these novel genes may help to elucidate their roles in facilitating adaptations of the uterus to pregnancy, including the expansion of myometrium in the pregnant uterus, and the maintenance of uterine quiescence during the majority of gestation. These processes have historically been among the least well understood in human physiology; however, the genomic approach of the present study has provided new insight into their molecular basis. We also speculate that pathological regulation of these gene products during pregnancy may result in pregnancy loss or preterm labour.
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Acknowledgements |
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The authors wish to thank Louella Hupp for editorial assistance. This work was supported by National Institutes of Health grant numbers HD11149 and T32 HD07190. Dedicated to Yasmin and Layla Rehman.
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