ABSTRACT
Early placental development is critical for successful pregnancy. Recently, we have reported that ~70 genes were differentially expressed in human placental villi between 6- and 8- weeks of gestation in cDNAbased expression arrays for ~400 PCR products, of which six specific gene products (COL4A4, CXCR4, ERBB2, HDAC1, HPRT1, and TNFRSF1A) appeared intriguing. In the present study we have examined expressions of these six candidate genes in placental villi obtained from 6-weeks, 7-weeks and 8-weeks (n = 6 for each group) human placental samples using quantitative real time RTPCR. We observed that there was considerable concordance (>95% confidence) in pair-wise analysis of transcript profiles between the two methods, however, absolute quantitative values as measured by quantitative RTPCR differed from those obtained from cDNA-based array analysis for 2 gene products (CXCR4 and ERBB2) out of 6 genes. No significant change was observed in the steady state expression of COL4A4 and HPRT1 during the time period examined. However, there was significant decrease in CXCR4 for 7-weeks (P< 0.01) and 8-weeks (P<0.05) samples, and significant (P<0.05) increase was seen for ERBB2 in 7-weeks and 8-weeks as compared to 6-weeks samples with no change between 7-weeks and 8-weeks samples. Moreover, significant (P< 0.05) increase for HDAC1 and decrease for TNFRSF1A was observed in 8-weeks samples as compared to 6-weeks samples with no change observed between 6-weeks and 7-weeks samples. We infer that it is essential that cDNA array-based data are verified in terms of quantitative estimates preferably by quantitative PCR before their use for any exploratory purpose. Taking together our previous array based data and the present study we conclude that a categorical balance exists between the expression of ERBB2 and HDAC1 genes affecting cell proliferation and differentiation in one hand, and CXCR4 and TNFRSF1A affecting chemotaxis, inflammatory response and apoptosis on the other hand. The expression of these genes appear important for the early development of human placental villi.
ABSTRACT
Human placental trophoblastic mass grows rapidly between 4 and 8 weeks of gestation making it highly vulnerable to external and internal challenges, however, there has been no reported study exploring the developmental molecular characteristics in human first trimester placental villi. In the present study, transcript expressions of human placental villi of normal pregnancies during 6 (n=6), 7 (n=6) and 8 (n=6) weeks of gestation using custom-tailored cDNA-based expression arrays for ~400 annotated human gene products were examined. Unsupervised and supervised analyses of expression data revealed that 386 (95%) genes were overtly involved in the first trimester placental villi, and these genes segregated into three clusters specifically corresponding to 6-, 7- and 8- weeks of gestation in principal component analysis. Bayesian prediction analysis based on relative expression levels of genes studied identified that expression patterns in 15 samples out of 18 samples showed concordance with high (0.8-1.0) confidence measures with the chronological age of the placenta, however, two samples collected during 7-weeks of gestation and one sample collected during 8-weeks of gestation were predicted to be 6- weeks sample with confidence measures between 0.6 and 0.5. Unsupervised hierarchical clustering analysis segregated the samples into two major branches; while one of them was composed of five 7-weeks samples only, the second major branch had three sub-branches: one of them was exclusively composed of three 8-week samples only, while other two subbranches were mainly composed of 6-weeks samples. K-means clustering analysis identified four optimal clusters of genes depending on the similarity of their relative expression for the set of genes studied across all the samples. Gene ontology (GO) based functional classifications of genes in K-means clusters revealed that the overall putative functions of co-regulated gene clusters were mutually comparable, however, specific genes related to ion homeostasis, metabolism, and VEGF activity specifically clustered in 8-weeks samples. Analysis of relative gene expression during in 6-8 weekplacental villi revealed that a large number of gene products were over represented by their either up-regulation (70 genes: ~18%) or down regulation (53 genes; ~14%) between 6 and 8 weeks villi samples and these genes are reportedly involved in biological processes like regulation of cell growth and proliferation, anti-apoptosis, angiogenesis, immune and inflammatory responses, extracellular matrix remodeling and multicellular organismal development involving almost all cellular components and molecular functions like signal transduction activity, transcription factor activity, nucleotide and protein binding, ion (especially calcium and zinc) binding and growth receptor activities. Interestingly, four genes (oxytocin receptor, tenascin C, TNF-R1 and retinol binding protein 1) showed differential regulation in human placental villi during 6-8 weeks of gestation, suggestive of an underlying network of regulation involving these factors in the developing placenta. To our knowledge, this is the first report indicating that these genes are involved in the early stage development of human placenta.