Analysis Of The Clonality Of Endometriotic Lesions Using HUMARA And PGK Gene Polymorphism Assay And Laser Capture Microdissection

Objective1. To explore the optimized strategy for the analysis of the clonality of endometriotic lesions using androgen receptor gene and phosphoglycerate kinase gene polymorphism and laser capture microdissection (LCM).2. To investigate the clonal pattern of endometriotic lesions with variable pathological subtypes.3. To investigate the distribution of monoclonal area for the epithelial cells of endometriotic gland.4. To investigate the clonal pattern of stromal cells of endometriotic lesions.Methods1. The specimens of endometriotic lesions used in this study included 5 frozen samples, 20 sample of archived formalin-fixed paraffin embedded tissue, and 2 samples of laser microdissected tissue. The observation and comparison for the gene amplifying efficiency were performed between the groups of fresh tissue and paraffin embedded tissue, the groups of with or without enzyme digestion of DNA with methylation-sensitive restriction endonuleases, the groups of normal PCR and nested PCR, the groups of with or without using whole genomic amplification for the DNA extracted for tissues. The flurescence labeling and gene fragment analysis were also applied for the clone detection.2. From 8 specimens of fresh endometriotic lesions with variable pathological subtypes, 50 samples of epithelial cells of isolated glands were micro-dissected and collected. The clonal analysis was performed for each gland by using the markers of AR and PGK gene polymorphism.3. From 20 samples of archived paraffin-embedded tissue of abdominal endometriosis, 94 samples of epithelial cells of isolated gland were micro-dissected and collected. The distribution and area of each clonal patch were determined by the inactivation pattern of HUMARA and PGK gene alleles.4. From 6 sample of abdominal endometriotic lesions, the stromal cells around multiple glands were micro-dissected and collected, and were underwent HUMARA and PGK polymorphism assay. Results1. The combined application of WGA and nested PCR significantly improved the amplificatin efficiency and increased the positive result during the clonal assay for limiting DNA, eg. micro-dissected cells. The combined enzyme digestion assay by using two methylated-sensitive restriction endonuclease, Hhaâ… and Hpaâ…¡, increased the proportion of informative samples. And using HUMARA and PGK as the associated marker minimized the non-informative cases with homozygous alleles in either gene. Additinally, HUMARA assay was adapted taking the advantage of an automated sequencer poviding high resolution of alleles and immediate quantitaion.2. Of 50 glands collected from fresh endometriotic tissues with variable pathological subtypes,38 glands were informative and showed monoclonal pattern for each, which implied that the epithelial cells from isolated gland probably originated from single progenitor cell. Moreover, the different subtype endometriotic lesions showed distinct clonal pattern, for instance, the glands within the lesions of ovarian endometriosis and deep infiltrating endometriosis exhibited uniform clonal pattern, while the peritoneal and abdominal wall endometriosis manfested as distinguishing pattern among different glands.3. Of 94 glands collected from archived paraffin-embedded endometriotic samples,76 glands were informative for gene assay and showed monoclonal pattern for each gland. The identical monoclonal pattern presented in between adjacent glands, while the divergent pattern could be found between the glands distant from each other. The glands located within the area of 0.01-0.25mm2 might share the identical monoclonal pattern and might grow from single stem cell.4. Of 6 samples of stromal cells around the endometriotic glands,3 presented as monoclonal pattern which was parallel with surrounding glands; while, the other 3 were identified as polyclonal pattern. This finding indicated that the stromal and epithelial cells in endometriotic foci might come from the same progenitor cell occasionally.Conclusions1. The combined gene amplification technique of whole genomic amplification and nested PCR, both the AR and PGK polymorphism assay, and automated fragment sequencing were the optimized protocol for clonality analysis with limiting samples obtained by laser capture microdissection. 2. The isolated gland of endometriotic lesions might originate from single precursor cell. A single lesion of ovarina endometriosis or deep infiltration lesion might derived from a single progenitor cell. While, the lesion of peritoneal or abdominal wall endometriosis seemed to be multicellular in origin.3. The clonal expansion of endometriotic precusor cell could form the lesion patch with size of about 0.25mm24. The stromal cells could share the common precusor cell with the surrounding glandular epithelial cells.