Large scale analysis of transcripts in human endometrium

Objective: We aimed to identify genes in normal human eutopic endometrium which respond to ovarian steroid hormone stimulation. We identify factors of possible interest in the pathogenesis of endometriosis.Design: The growth of ectopic endometrium (endometriosis) is the consequence of the complex interaction of many genes within endometrium and the tissues at the sites of implantation. Many factors and signalling pathways are involved in this pathophysiological process. The ovarian steroids in particular, are known to play a key role in the proliferation and differentiation of both normal and ectopic endometrium. As a first step towards identifying the endometrial genes that respond to ovarian steroids and which therefore may be important in the pathogenesis of endometriosis, we have carried out large-scale analysis of all the mRNAs in normal human endometrium from both proliferative and secretory phases of the menstrual cycle.Materials/Methods: High density, gene microarrays allow the simultaneous and quantitative analysis of the expression of thousands of genes, and hence provides a broader picture of gene-gene interactions than previously available using conventional techniques. Using a high density oligonucleotide array (Affymetrix Inc., USA), which comprises 60,000 known gene and EST sequences, we determine “total” gene expression in proliferative and secretory endometrium. Five human endometrial biopsies were obtained from both phases of the cycle; days 9 to 11 in the proliferative phase and days LH + 6 to 8 (as judged by a urinary assessment of the LH surge) for the secretory phase. Total RNA was extracted and a biotin-labelled cRNA target prepared which was then hybridised to the Genechips. Images were scanned and the hybridisation levels determined for all 60,000 transcripts on the chips.Results: The abundance and differences in transcript levels between proliferative and secretory phase endometrium have been analysed for the 12,000 most well defined genes. Local variance values were used to define the range limits for this population of genes, and a total of 34 gene sequences were identified as having highly significant differences (i.e. were outside of the 99.99% range limits). Of these 34 transcripts, 24 were shown to be upregulated in the secretory vs. proliferative phase endometrium. As expected, this includes transcripts such as placental protein 14 and osteopontin that have previously been documented to be progesterone responsive. The remaining 10 genes were upregulated in the proliferative vs. secretory phase endometrium. This group contained previously recorded oestrogen responsive transcripts such as stromelysin. In addition, several novel genes were found to be highly expressed in human endometrium and also either oestrogen or progesterone responsive. Examination of the 5′ promotor regions of these genes identified many oestrogen and progesterone receptor DNA and transcription factor binding domains that will be the subject of the presentation.Conclusions: In conclusion, we have identified a subset of genes that were previously not known to be differentially regulated in human endometrium, most likely in response to oestrogen and progesterone, which may have implications for endometrial physiology and potentially pathophysiology.Supported By: BBSRC and Pfizer Ltd. Objective: We aimed to identify genes in normal human eutopic endometrium which respond to ovarian steroid hormone stimulation. We identify factors of possible interest in the pathogenesis of endometriosis. Design: The growth of ectopic endometrium (endometriosis) is the consequence of the complex interaction of many genes within endometrium and the tissues at the sites of implantation. Many factors and signalling pathways are involved in this pathophysiological process. The ovarian steroids in particular, are known to play a key role in the proliferation and differentiation of both normal and ectopic endometrium. As a first step towards identifying the endometrial genes that respond to ovarian steroids and which therefore may be important in the pathogenesis of endometriosis, we have carried out large-scale analysis of all the mRNAs in normal human endometrium from both proliferative and secretory phases of the menstrual cycle. Materials/Methods: High density, gene microarrays allow the simultaneous and quantitative analysis of the expression of thousands of genes, and hence provides a broader picture of gene-gene interactions than previously available using conventional techniques. Using a high density oligonucleotide array (Affymetrix Inc., USA), which comprises 60,000 known gene and EST sequences, we determine “total” gene expression in proliferative and secretory endometrium. Five human endometrial biopsies were obtained from both phases of the cycle; days 9 to 11 in the proliferative phase and days LH + 6 to 8 (as judged by a urinary assessment of the LH surge) for the secretory phase. Total RNA was extracted and a biotin-labelled cRNA target prepared which was then hybridised to the Genechips. Images were scanned and the hybridisation levels determined for all 60,000 transcripts on the chips. Results: The abundance and differences in transcript levels between proliferative and secretory phase endometrium have been analysed for the 12,000 most well defined genes. Local variance values were used to define the range limits for this population of genes, and a total of 34 gene sequences were identified as having highly significant differences (i.e. were outside of the 99.99% range limits). Of these 34 transcripts, 24 were shown to be upregulated in the secretory vs. proliferative phase endometrium. As expected, this includes transcripts such as placental protein 14 and osteopontin that have previously been documented to be progesterone responsive. The remaining 10 genes were upregulated in the proliferative vs. secretory phase endometrium. This group contained previously recorded oestrogen responsive transcripts such as stromelysin. In addition, several novel genes were found to be highly expressed in human endometrium and also either oestrogen or progesterone responsive. Examination of the 5′ promotor regions of these genes identified many oestrogen and progesterone receptor DNA and transcription factor binding domains that will be the subject of the presentation. Conclusions: In conclusion, we have identified a subset of genes that were previously not known to be differentially regulated in human endometrium, most likely in response to oestrogen and progesterone, which may have implications for endometrial physiology and potentially pathophysiology. Supported By: BBSRC and Pfizer Ltd.