Microarray analysis of differentially expressed genes in placental tissue of pre-eclampsia: up-regulation of obesity-related genes

Molecular Human Reproduction Vol.8, No.7 pp. 674–680, 2002 Microarray analysis of differentially expressed genes in placental tissue of pre-eclampsia: up-regulation of obesityrelated genes T.Reimer1, D.Koczan, B.Gerber, D.Richter, H.J.Thiesen and K.Friese Department of Obstetrics and Gynaecology and Institute of Immunology, University of Rostock, Germany 1To 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: 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. Key words: leptin/microarray/obese gene/pre-eclampsia Introduction 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). 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 674 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 highdensity 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. Materials and methods 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 preeclampsia (n ⫽ 6; 艋32 weeks gestation). Pre-eclampsia was defined as a blood pressure of 艌140/90 mmHg taken twice, 6 h apart, with proteinuria of 艌2⫹ or 艌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 艌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. © European Society of Human Reproduction and Embryology Obesity-related genes in pre-eclampsia Table I. Clinical data of the 12 included patients. The mRNAs were pooled from the six patients of each group to normalize for individual variation. No chorioamnionitis was observed in any of the cases History Age (years) Normotensive group G1P0 25 G2P0 25 G1P0 21 30 G3P1 19 G1P0 G7P3 32 Pre-eclamptic group 25 G1P0 20 G1P0 G1P0 31 G1P0 29 G1P0 29 G1P0 24 Gestation (week) Birth weight (g) Betamethasone Pathogenesis Mode of delivery IUGR UA pH Apgar score 32 32 25 32 32 31 1970 1890 900 1800 1900 1550 o o ⫹ ⫹ ⫹ o Sp.lab. PROM Sp.lab. PROM Sp.lab. PROM Vaginal Vaginal Caesarean Vaginal Vaginal Caesarean o o o o o o 7.32 7.43 7.17 7.31 7.28 7.31 8/10/10 7/7/7 2/4/5 7/7/8 8/9/9 2/3/6 31 32 29 29 32 30 1460 1320 940 750 1440 1110 o ⫹ ⫹ ⫹ o ⫹ Pre-ecl. Pre-ecl. Pre-ecl. Pre-ecl. Pre-ecl. Pre-ecl. Caesarean Caesarean Caesarean Caesarean Caesarean Caesarean o o ⫹ ⫹ o o 7.37 7.22 7.34 7.28 7.14 7.25 5/7/8 4/8/7 3/6/8 2/6/6 7/8/8 2/6/7 IUGR ⫽ intrauterine growth retardation; UA pH ⫽ pH value in umbilical artery; Sp.lab. ⫽ spontanous labour; PROM ⫽ premature rupture of the membrane. 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 a (...truncated)