ANALYSIS OF CLONALITY IN HUMAN ENDOMETRIOTIC CYSTS BASED ON EVALUATION OF X-CHROMOSOME INACTIVATION IN ARCHIVAL FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE

The clonality of epithelial cells in ovarian endometriotic cysts was e valuated on the basis of a polymorphism of the X-linked phosphoglycera te kinase gene (PGK). The problems associated with clonality analysis of DNA samples extracted from archival formalin-fixed, paraffin-embedd ed tissue were addressed with the use of newly designed primers and a modified stepdown program for PCR amplification. The method relies on digestion of DNA with the methylation-sensitive restriction enzyme Sna Bl, PCR amplification of PGK, and detection of a BstXl polymorphism. W ith this approach, only the inactive (methylated) PGK allele is select ively amplified by PCR. A total of 25 epithelial cells in endometrioti c cysts and 25 matched normal ovarian stromal tissue specimens were an alyzed. All of the control tissue samples yielded PCR products, with 1 1 of the 25 samples found to be heterozygous for the BstXl polymorphis m. Ten of the 25 epithelial cells in endometriosis specimens were info rmative at this locus, and all of these 10 were shown to be monoclonal on the basis of PGK methylation. The results indicate that this new m ethod of clonal analysis with archival formalin-fixed tissue is reliab le and confirm that endometriotic cysts are monoclonal in origin.