Up-regulation of endocrine gland-derived vascular endothelial growth factor but not vascular endothelial growth factor in human ectopic endometriotic tissue  Kai-Fai Lee, Ph.D., Yin-Lau Lee, Ph.D., Rachel W.S. Chan, Ph.D., Ana W.Y. Cheong, M.Sc., Ernest H.Y. Ng, M.D., Pak-Chung Ho, M.D., William S.B. Yeung, Ph.D.  Fertility and Sterility  Volume 93, Issue 4, Pages 1052-1060 (March 2010) DOI: 10.1016/j.fertnstert.2008.12.001 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Real-time polymerase chain reaction analysis of endocrine gland-derived vascular endothelial growth factor (EG-VEGF), prokineticin receptor 1 (PKR1), and prokineticin receptor 2 (PKR2) mRNA expression levels between normal and eutopic endometrium. Eutopic endometrial samples from normal women (NE) in proliferative (Proli, n = 14) and secretory phases (Sec, n = 19) and from women with endometriosis (DE) in proliferative (n = 9) and secretory phases (n = 6) were studied. The relative expression of the (A) EG-VEGF, (B) PKR1, and (C) PKR2 mRNA expressions were normalized to 18S expression and calculated by the equation: 2-ΔΔCt using the proliferative phase normal endometrium as calibrator. A difference of threshold cycle (Ct) values (ΔCt) as obtained by subtracting the Ct value of 18S from the Ct value of the genes of interest. The relative gene expression values were calculated by the 2-ΔΔCt method (see Materials and Methods section). Data were presented as median with 10th, 25th, 75th, and 90th percentiles and outliers (•). Statistical analysis was performed by Kruskal-Wallis test and P values were shown. A P value less than .05 was considered significantly different from different samples. Fertility and Sterility 2010 93, 1052-1060DOI: (10.1016/j.fertnstert.2008.12.001) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Real-time polymerase chain reaction analysis of endocrine gland-derived vascular endothelial growth factor (EG-VEGF), prokineticin receptor 1 (PKR1), and prokineticin receptor 2 (PKR2) mRNA expression levels between normal and eutopic endometrium. Eutopic endometrial samples from normal women (NE) in proliferative (Proli, n = 14) and secretory phases (Sec, n = 19) and from women with endometriosis (DE) in proliferative (n = 9) and secretory phases (n = 6) were studied. The relative expression of the (A) EG-VEGF, (B) PKR1, and (C) PKR2 mRNA expressions were normalized to 18S expression and calculated by the equation: 2-ΔΔCt using the proliferative phase normal endometrium as calibrator. A difference of threshold cycle (Ct) values (ΔCt) as obtained by subtracting the Ct value of 18S from the Ct value of the genes of interest. The relative gene expression values were calculated by the 2-ΔΔCt method (see Materials and Methods section). Data were presented as median with 10th, 25th, 75th, and 90th percentiles and outliers (•). Statistical analysis was performed by Kruskal-Wallis test and P values were shown. A P value less than .05 was considered significantly different from different samples. Fertility and Sterility 2010 93, 1052-1060DOI: (10.1016/j.fertnstert.2008.12.001) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Real-time polymerase chain reaction analysis of endocrine gland-derived vascular endothelial growth factor (EG-VEGF), prokineticin receptor 1 (PKR1), and prokineticin receptor 2 (PKR2) mRNA expression levels between normal and eutopic endometrium. Eutopic endometrial samples from normal women (NE) in proliferative (Proli, n = 14) and secretory phases (Sec, n = 19) and from women with endometriosis (DE) in proliferative (n = 9) and secretory phases (n = 6) were studied. The relative expression of the (A) EG-VEGF, (B) PKR1, and (C) PKR2 mRNA expressions were normalized to 18S expression and calculated by the equation: 2-ΔΔCt using the proliferative phase normal endometrium as calibrator. A difference of threshold cycle (Ct) values (ΔCt) as obtained by subtracting the Ct value of 18S from the Ct value of the genes of interest. The relative gene expression values were calculated by the 2-ΔΔCt method (see Materials and Methods section). Data were presented as median with 10th, 25th, 75th, and 90th percentiles and outliers (•). Statistical analysis was performed by Kruskal-Wallis test and P values were shown. A P value less than .05 was considered significantly different from different samples. Fertility and Sterility 2010 93, 1052-1060DOI: (10.1016/j.fertnstert.2008.12.001) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Real-time polymerase chain reaction analysis of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and vascular endothelial growth factor (VEGF) mRNA expression levels in paired laser-captured microdissection isolated eutopic and ectopic endometriotic samples. (A) Three laser-captured microdissection sections from ectopic or eutopic samples were used for RNA extraction. (B) The relative expressions of EG-VEGF and VEGF transcripts were normalized to 18S and calculated by the equation: 2-ΔΔCt using the ectopic endometrium as calibrator. A P value less than .05 was considered significantly different from different samples. Ct = threshold cycle; NS = not significant. Fertility and Sterility 2010 93, 1052-1060DOI: (10.1016/j.fertnstert.2008.12.001) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Immunohistochemical staining of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) in eutopic and ectopic endometrium. Frozen eutopic (A, C) and ectopic (B) endometrial, and archive testis (D) samples were stained with EG-VEGF (A, B, D) antibody. Specific brown signal was found in the glandular (GE) and luminal epithelium (LE) of eutopic endometrium (A), and in the stromal cells (SC) of the ectopic endometrium (B) of the same patient and no signal was found when the primary antibody was omitted (C). Leydig cells (Le) of frozen human testicular sample were positively stained with EG-VEGF antibody, but not in spermatozoa (Sp) in the seminiferous tubule (D). The expression of EG-VEGF protein in 12 pairs of endometriotic samples was determined by H scoring. Significant difference was found in the stromal cells (E), but not glandular epithelium (F) of ectopic and eutopic endometrium (P=.011). Fertility and Sterility 2010 93, 1052-1060DOI: (10.1016/j.fertnstert.2008.12.001) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions