Molecular Human Reproduction, Vol. 8, No. 7, 674-680,
July 2002
© 2002 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Microarray analysis of differentially expressed genes in placental tissue of pre-eclampsia: up-regulation of obesity-related genes
Department of Obstetrics and Gynaecology and Institute of Immunology, University of Rostock, Germany
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Abstract |
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Susceptibility genes present in both mother and fetus most likely contribute to the risk of pre-eclampsia. Placental biopsies were therefore investigated by high-density DNA microarray analysis to determine genes differentially regulated within chorionic villous tissue in pre-eclampsia and normal pregnancy. The pooled RNAs of pre-eclamptic and normotensive subjects were hybridized to the HuGeneFL array representing sequences from
5600 full-length human cDNAs. The differentially expressed genes that were detected could be categorized into nine groups: adhesion molecules, obesity-related genes, transcription factors/signalling molecules, immunological factors, neuromediators, oncogenic factors, protease inhibitors, hormones and growth factor-binding proteins. Among those, the obesity-related genes included putative candidate genes associated with the pathogenesis of pre-eclampsia. One of the most up-regulated transcripts was the obese gene (43.6-fold change), and this was reflected by elevated leptin protein levels. In the case of feto-maternal contribution of polymorphic genes to pre-eclampsia, expression analysis of placental tissue has lead to numerous target genes waiting for large scale genetic linkage analyses. leptin/microarray/obese gene/pre-eclampsia
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Pre-eclampsia is a major cause of maternal and fetal morbidity and mortality, affecting 3–5% of all pregnancies. Manifestations include increased blood pressure and proteinuria, and
30% of fetuses born to pre-eclamptic women are less than the tenth percentile for weight and are considered growth restricted. Although the aetiology remains to be elucidated, the placenta is undoubtedly involved in the pathogenesis of pre-eclampsia, since termination of pregnancy eradicates the disease (Roberts and Cooper, 2001
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Susceptibility genes from both mother and fetus may contribute to the risk of pre-eclampsia. For instance, genomic imprinting controls the expression of maternally or paternally derived genes expressed in fetal cells. Inheritable paternal, rather than maternal, imprinting of particular genes is necessary for normal development of trophoblast and extra-embryonic membranes (Dekker and Sibai, 2001). In particular, data based on a Norwegian study clearly demonstrate the impact of paternal factors on the risk of developing pre-eclampsia (Lie et al., 1998). A recent study described that men and women who were the product of a pregnancy complicated by pre-eclampsia were significantly more likely than those without such a history to have a child who is also the product of a pregnancy complicated by pre-eclampsia (Esplin et al., 2001). These findings support the hypothesis that the genotype of the fetus contributes to the overall risk of pre-eclampsia.
The applications of DNA microarrays are ideal for studies of genomic structure (e.g. mutation and polymorphism analyses) and for monitoring gene expression (Bilban et al., 2000). The objective of the present study was to examine the critical events in trophoblast tissue underlying development of pre-eclampsia at a genomic level. Different placental biopsies were taken to be investigated by high-density DNA microarray analysis since the placenta is most severely affected in the early stages of pre-eclamptic pathophysiology, possibly due to incomplete invasion of fetal trophoblast cells into the uterus.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Human subjects
The study was conducted at the Department of Obstetrics and Gynaecology, University of Rostock, Germany, and was approved by the Institutional Review Board. Placental biopsies were obtained during Caesarean section or after vaginal delivery from both normotensive patients and those with pre-eclampsia (n = 6;
32 weeks gestation). Pre-eclampsia was defined as a blood pressure of 
140/90 mmHg taken twice, 6 h apart, with proteinuria of 2+ or 300 mg in a 24 h collection. Normotensive subjects (n = 6) were matched for maternal and gestational ages, and for pre-pregnancy body mass index; however, pre-eclamptic women delivered newborns with lower birth weight percentiles (Mann-Whitney U-test; P = 0.002). Intrauterine growth retardation (IUGR) was defined as birth weight below the third percentile for gestational age.
The gestational age was calculated from day 1 of the last menstrual period, unless ultrasonography results demonstrated a discrepancy of 14 days, in which case ultrasonographic dating of the pregnancy was used for calculation. Patients with IUGR showed sonographic signs of decreased amniotic fluid volumes and changes in umbilical artery blood flow. Information on maternal reproductive data, labour and delivery characteristics, and infant outcomes were collected from maternal medical records and are presented in Table I.
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Plasma samplings
After informed consent was given, venous blood samples were obtained from the women at time of admission to the case room. Blood samples from the umbilical vein were taken at birth. All samples were centrifuged at 2000 g for 15 min at 4°C. Plasma was collected and stored at –20°C until the assay was performed.
Tissue preparation
Human placentae were obtained after vaginal delivery or Caesarean section. A defined central chorionic tissue area was dissected and the maternal decidua and amnionic membrane were removed. Tissues were frozen and stored in liquid nitrogen until use.
RNA extraction
Total RNA was prepared from the chorionic villous tissues with a RNeasy mini-kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. The quality of the RNA samples was determined by electrophoresis through denaturing agarose gels and staining with ethidium bromide. The RNA was quantified and evaluated for purity by UV spectrophotometry. To further assess the quality of the RNA, all specimens were tested by analysis of housekeeping gene expression using conventional RT–PCR.
Microarray analysis
Despite the power of `high through-put' technologies to show general patterns of gene expression, variations in gene expression among individuals must also be considered. One potential approach is to pool mRNA from 5–10 people from each group to normalize for individual variation (Cristofalo, 2000). We therefore pooled the six patients of each group and the quality of pooled mRNAs were checked with GeneChip expression analysis Test-2 probe array (Affymetrix, Santa Clara, CA, USA).
Following mRNA isolation and pooling, mRNAs were labelled and hybridized to the HuGeneFL array (Affymetrix) representing sequences from 5600 genes using >7000 full-length human cDNAs according to the manufacturer's instructions. Arrays were scanned using an Affymetrix confocal scanner and analysed using GeneChip 3.0 software (Affymetrix). According to the literature, the minimum detectable fold change for differential expression is 1.4 (Yue et al., 2001). We have chosen a threshold of 3.0 to exclusively demonstrate significant changes in global mRNA expression.
TaqMan real-time RT–PCR
To confirm the manufacturer's data for selected genes, we used quantitative real-time RT–PCR analysis (ABI PRISM 7700 Sequence Detection System; Perkin-Elmer, Foster City, CA, USA). This novel approach has been described in detail previously (Reimer et al., 2000). Briefly, during the extension phase of the PCR, the polymerase cleaves the fluorescent-labelled TaqMan probe, resulting in a release of the reporter dye. The algorithm normalizes the reporter signal to a passive reference. Next, the algorithm multiplies the SD of the background reporter signal in the first few cycles (cycles 3–15) by a default factor of 10, to determine a threshold. The cycle at which this baseline level is exceeded is defined as the threshold cycle (Ct). The Ct values of the samples depend on the initial template copy number.
The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize mRNA concentration. The TaqMan GAPDH control reagents (Perkin-Elmer) provided the necessary components including primer pairs and TaqMan probe. The following primers and TaqMan probes were designed using exon/intron junctions to avoid amplification of DNA sequences: leptin: forward, 5'-CCAGGATCAATGACATTTCACA; reverse, 5'-GAATGAAGTCCAAACCGGTG; TaqMan probe, 5'-ACGCAGTCAGTCTCCTCCAAACAGAAA. Integrin 
1: forward, 5'-CACCATTGTTAAAACTCTGGGA; reverse, 5'-CAAATGAAGCTGCTGACTGG; TaqMan probe, 5'-TTGCCCTGGAAGCCACAGCTG. Primers and probes were obtained from Applied Biosystems GmbH (Weiterstadt, Germany). Commercial reagents (TaqMan EZ RT–PCR Kit; Perkin-Elmer) and conditions were applied according to the manufacturer's protocol. All RT–PCR reactions were performed in triplicate with a final volume of 25 µl. The thermocycler parameters were 2 min at 50°C, 30 min at 60°C and 1 min at 95°C for RT, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C for PCR amplification.
Immunohistochemistry
For the immunohistochemical characterization, cryosections from placental tissue were examined. All samples (n = 10) were fixed in 3.7% buffered formalin and in pure methanol. Sections 8 µm thick were immunostained by the diaminobenzidine (DAB) procedure using automated NexES Special Stain System (Ventana Medical Systems, Tucson, AZ, USA). The slides were incubated in either polyclonal antibody raised in rabbits against human leptin (A-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), at a dilution of 1:200 in phosphate-buffered saline (PBS), for 32 min at 37°C; or mouse monoclonal antibody against human integrin 1 (Endogen, Woburn, MA, USA), at a dilution of 1:50 in PBS, for 32 min at 37°C. The sections were counterstained with haematoxylin for 2 min. The stained slides were evaluated by the department's pathologist. Immunohistochemical results were analysed by light microscopy and digital images were obtained with an Olympus DP11 digital-camera system (Olympus Optical Co., GmbH, Hamburg, Germany).
Hormone assay
Human leptin levels in maternal and umbilical cord plasma samples were determined by use of an ELISA for human leptin (Diagnostic Systems Laboratories, Webster, TX, USA).
Statistical analysis
The statistical test performed was the Mann-Whitney U-test using Statistics Package for Social Sciences for Windows, Release 8.0 (SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant. All P-values cited were from two-tailed tests.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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By comparative differential gene expression analysis, 59 genes were found to be significantly altered in their expression levels by
3-fold: 44 genes were found to be up- and 15 genes to be down-regulated in pre-eclampsia (Table II). These genes were assigned to nine functional subsets: adhesion molecules, obesity-related genes, transcription factors/signalling molecules, immunological factors, neuromediators, oncogenic factors, protease inhibitors, endocrine-related proteins and growth factor-binding proteins. Among those, several genes involved in the regulation of the obese gene were found to be up-regulated in pre-eclampsia (Figure 1).
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The transcript of the placental obese gene (placental leptin) itself was up-regulated in our pre-eclamptic samples by a change of 43.6-fold (Table II
). Leptin protein levels were therefore determined in umbilical cord blood and corresponding prepartal maternal serum samples. Significant differences in leptin concentrations (P = 0.017) were detected in maternal serum between pre-eclamptic (median: 59.0 mg/l; range: 40.2–59.9) and normotensive patients (35.5 mg/l; 22–55). Although absolute leptin values were found to be decreased in cord plasma compared with maternal samples, these differences remained significant (P = 0.016) between pre-eclamptic (6.7 mg/l; 4.3–9.0) and normotensive cases (1.4 mg/l; <0.5–2.3). Thus, increased obese gene mRNA levels originally found by RNA profiling corresponded to elevated protein levels in pre-eclampsia.
To verify the microarray data for the most up-regulated genes, we determined placental integrin 1 and leptin expression at mRNA and protein levels using other approaches. In general, the microarray data of the selected genes were confirmed by TaqMan real-time RT–PCR and immunohistochemistry. The placental tissue of patients with pre-eclampsia showed a significantly higher leptin mRNA expression than the normotensive group (P = 0.002). In placental tissue of patients with pre-eclampsia, there was also a higher integrin 1 mRNA expression than in placentas derived from normotensive patients. However, this difference was not statistically significant (Table III).
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By immunohistochemistry, all five pre-eclamptic samples studied showed a moderate immunoreactivity for leptin, whereas samples from the normotensive group (n = 5) revealed weak immunostaining patterns (Figures 2A,B
). The villi presented intense immunoreactivity for leptin in the villous mesenchyme. No positivity was found on the outer areas, i.e. in the cytoplasm of syncytiotrophoblast or trophoblast cells, and in stromal vessels. Immunohistochemical staining of integrin 1 showed a strong expression in all pre-eclamptic samples and a moderate (n = 3) to strong (n = 2) expression in chorionic villi derived from normotensive patients. Integrin 1 immunoreactivity was found in the villous mesenchyme, but to a lesser extent in the outer areas, i.e. in the cytoplasm of syncytiotrophoblast or trophoblast cells, or in the endothelial cells of stromal vessels (Figures 3A,B).
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Especially for leptin, the semi-quantitative analysis of immunohistochemical findings supported the detected mRNA results. However, the comparison of the two techniques used is limited since the TaqMan RT–PCR analysis quantitates mRNA transcript numbers while immunohistochemical studies deliver more qualitative data on protein expression. Thus, the real-time RT–PCR analysis confers higher sensitivities and accuracies compared with immunohistochemical stainings. Furthermore, the sensitivity of the immunohistochemical data might vary depending on the binding affinities of antibodies used.
A problem of the presented study is the unavailability of a normal control group. The matched normotensive patients had other pathological reasons for delivering prematurely including early spontaneous labour or preterm premature rupture of the membranes. The majority of pre-eclamptic patients (n = 5; 83%) delivered by Caesarean section without previous uterine activity, whereas this type of delivery occurred with only one patient in the normotensive group (17%). These are, in fact, not ideal controls, but a substitute, since placental biopsies cannot be obtained from intact normal pregnancies 32 weeks gestation delivered by elective Caesarean section. However, interactions between chorioamnionitis or betamethasone and the detected regulated RNA transcripts are unlikely, since no chorioamnionitis was observed in all cases and the frequency of using betamethasone for fetal lung maturation was not different between the groups (Table I).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Our microarray approach revealed that components of cell adhesion- (i.e. integrin
1) and obesity-related genes (i.e. leptin) are considerably up-regulated in pre-eclampsia. However, microarray results can be influenced by each step of the complex assay, from array manufacturing to sample preparation (extraction, labelling, hybridization) and image analysis. The efficiency of the RT reaction is known to be affected by the enzyme, primers, nucleotides and RNA secondary structure (Rajeevan et al., 2001). These factors in turn influence the representation of transcripts in the final cDNA probe, and necessitate the need for validations by complementary techniques. As microarrays tend to have a low dynamic range, which leads to small but significant under-representations of fold changes in gene expression, RT–PCR with a higher dynamic range is used more to validate the observed trends rather than duplicate the absolute values obtained by chip experiments. The described overall concordance of trends between the two techniques is 40–80% (Sgroi et al., 1999; Chaib et al., 2001; Rajeevan et al., 2001). As seen in Table III, the trends toward up-regulation of leptin and integrin 1 in pre-eclamptic samples observed by array analysis were consistent with those detected by real-time RT–PCR.
Several diseases, including pre-eclampsia and IUGR, may be explained by anomalies in integrin patterns (Merviel et al., 2001). Integrins are heterodimer glycoproteins (two subunits: and ß). Subunit regulates ligand binding and the extracellular confirmation, while the ß subunit has an intracellular domain associated with cytoskeleton proteins. The 1ß1 heterodimer (receptor for laminin and for type I and IV collagens) is involved in the trophoblast cell transition from a proliferative to an invasive phenotype (Pasqualini and Hemler, 1994). However, verification of integrin 1 microarray data (43.7-fold change) using real-time RT–PCR and immunohistochemistry showed only a marginal up-regulation of integrin 1 mRNA and protein in pre-eclampsia.
In contrast, the result of the cDNA microarray for leptin (43.6-fold change) was supported by RT–PCR, immunohistochemistry and ELISA analyses, indicating a key role for this obesity-related protein in the pathogenesis of pre-eclampsia. The positive immunostaining pattern for leptin in the villous mesenchyme of our preterm placental samples is a contrast to findings in normal mature placentae where leptin immunoreactivity has been shown to be present in the cytoplasm of syncytiotrophoblast cells, but not in the core of villi (Senariset al., 1997). The villous mesenchyme mainly consists of fibroblasts and Hofbauer cells (macrophages). According to the shape of the immunostained cells and counterstained nuclei, the fibroblasts are candidates for leptin synthesis. There has been evidence of leptin synthesis and secretion by human fibroblasts, and this is not unexpected as fibroblasts and adipocytes share a common stem cell origin (Glasow et al., 2001).
Increased leptin levels result in a negative energy balance (i.e. energy expenditure greater than food intake), whereas decreased levels lead to a positive energy balance due to the activities of the leptin receptor in the arcuate nucleus of the hypothalamus (Friedman, 2000). Leptin mRNA and the leptin receptor have previously been identified in mouse and human placenta, suggesting that leptin acts as a humoral signal from the placenta to the mother and fetus independent of body fat content (Friedman and Halaas, 1998). The increased leptin mRNA might be a consequence of the decreased placental perfusion in pre-eclampsia leading to hypoxic compartments of the organ. It is of interest that hypoxia increases leptin production in human BeWo choriocarcinoma cells (Mise et al., 1998).
The considerable elevation of placental leptin in pre-eclampsia is consistent with the pathophysiology of hypertension being mediated via known receptors present in vascular endothelial cell types, possibly due to a contribution of leptin to an increased central sympathetic tone (Tartaglia et al., 1995; Schobel et al., 1996; Narkiewicz et al., 1999). Leptin has also been implicated in regulating fetal growth and development by binding to leptin receptors present in fetal organs. The relevance of leptin protein in the pathophysiology of pre-eclampsia has previously been associated with significantly elevated leptin protein levels in cord plasma of preterm infants with IUGR and in those of pre-eclamptic mothers (Hytinantti et al., 2000).
In addition to its effects on blood pressure and body weight, leptin has a variety of other functions, including the regulation of immune responses. In fact, proinflammatory cytokines increase leptin levels, whereas leptin regulates the production of several pro- and anti-inflammatory cytokines. Leptin also modulates T-lymphocyte responses. It induces the production of large amounts of interferon (IFN)-
and interleukin (IL)-2, and decreases IL-4 production (Lord et al., 1998). Proinflammatory cytokines such as IFN- are known to up-regulate the expression of adhesion molecules, which are detectable in pre-eclamptic placental biopsies (Boehm et al., 1997).
It has been found that maternal plasma leptin concentrations are elevated before pre-eclampsia is clinically evident (Anim-Nyame et al., 2000), suggesting that the increased expression of obesity-related genes is more than an epiphenomen and that these genes are involved in the pathogenesis of pre-eclampsia. As shown in Figure 1, a group of obesity-related genes were found to be differentially expressed in pre-eclampsia. The obese gene promoter is responsive to several transcription factors, including C/EBP (3.0-fold change) and peroxisome proliferator-activated receptor- (1.5-fold change) which are up-regulated in pre-eclampsia (Friedman and Halaas, 1998). Cholecystokinin (3.1-fold change) belongs to the short-term system for controlling energy balance and potentiates the anorectic effect of leptin (Matson et al., 1997), whereas the secretion of pancreatic zymogen granule membrane protein GP-2 (5.2-fold change) is linked to cholecystokinin (Hoops et al., 1993). The increase in prolactin (6.4-fold change) and leptin early in pregnancy suggests an association between these two hormones (Mukherjea et al., 1999). The local production of prolactin exerts autocrine/paracrine actions to enhance implantation and early expansion of the blastocyst. In addition, prolactin regulates multiple functions of decidual immune cells involved in protecting the semiallogenic embryo from rejection since it is known that prolactin influences the response of immune cells (Tseng and Mazella, 1999).
To have the best chance of identifying genetic anomalies associated with pre-eclampsia, genetic studies should concentrate on women who develop pre-eclampsia in their first pregnancy (Broughton Pipkin, 1999). Assuming a maternal inheritance or fetal transmission of a paternal gene, our expression analysis of placental tissue in pre-eclamptic primigravidas identifies putative target genes for large scale genetic linkage analyses as well as for detailed single nucleotide polymorphism (SNP) studies of genes. Such a study, sampling from mother, father, newborn and mother's parents, is currently being founded by the British Heart Foundation as a 10-centre collaboration in the UK, the genetics of pre-eclampsia (GOPEC) study (Broughton Pipkin and Roberts, 2000). Altered gene expression in placental tissues detected by our microarray analysis in pre-eclampsia specify a gene set of paternal and/or maternal origin. Functional relevance of the gene in disease pathways might be due to specific combinations of gene modifications such as SNPs or methylation patterns displayed by maternal–fetal sharing of recessive gene(s) and/or dominant gene(s) with low penetrance.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We thank H.Terpe for immunohistochemical evaluation.
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Notes |
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1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, University of Rostock, P.O. Box 10 08 88, D-18055 Rostock, Germany. E-mail: toralf.reimer{at}med.uni-rostock.de
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