1. An epigenetic disorder may cause aberrant expression of aromatase gene in endometriotic stromal cells 
Masao Izawa, Ph.D., Tasuku Harada, M.D., Fuminori Taniguchi, M.D., Yoko Ohama, M.D., Yasuko Takenaka, M.D., Naoki Terakawa, M.D. 
Fertility and Sterility 
Volume 89, Issue 5, Pages (May 2008)
DOI: /j.fertnstert
Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
17β-Estradiol production in vitro in endometrial and endometriotic stromal cell cultures. Endometrial (six from the proliferative phase and six from the secretory phase donors) and endometriotic cells (10 from the follicular phase and eight from the luteal phase donors) were incubated in the presence of 5 × 10−6 M testosterone for 8 hours. At the end of incubation, the culture medium was removed, and the 17β-estradiol level was estimated using the enzyme immunoassay kit in duplicate. Mean values are expressed as pg/mL/10 5 cells. The variation in the intra-assay was less than 10%. a, b versus c, P<.001. a, b versus d, P <.001.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Aromatase mRNA expression in endometrial and endometriotic cells. (A) Stromal cells were collected from two endometrial (proliferative phase, no. 1, and secretory phase, no. 2) and three endometriotic tissues (one from follicular phase, no. 1, and the other two from luteal phase, nos. 2 and 3). Single-stranded cDNA was prepared from total cellular RNA and subjected to PCR. A sequence corresponding to the total open reading frame from Exon II to Exon X (1518 bp) was amplified and subjected to a 1.5% agarose gel electrophoresis. As an internal control, β-tubulin mRNA was assayed in parallel. The amplified signal was visualized using ethidium bromide staining under ultraviolet light. (B) Quantitative analysis of aromatase mRNA expression by real-time PCR. Single-stranded cDNAs from endometrial (five proliferative phase and five secretory phase) and endometriotic cells (six follicular and six luteal phase), which were randomly picked up from cells examined in Figure 1, were subjected to the TaqMan real-time PCR in triplicate. Values are expressed as arbitrary units. a, b versus c, P<.001. a, b versus d, P<.001. c vs. d, P<.001.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Effect of 5-Aza-dC-treatment on aromatase mRNA expression in endometrial cell cultures. Stromal cells collected from two endometrial tissues (proliferative phase) were treated with 5-Aza-dC at a concentration of 5 μM for 96 hours, and total cellular RNAs and the single-stranded cDNAs were prepared. (A) Aromatase mRNA expression. A sequence corresponding to the total open reading frame (1518 bp) was amplified and subjected to agarose gel electrophoresis. As an internal control, β-tubulin mRNA was assayed in parallel. Amplified Sequences were visualized by ethidium bromide staining. (B) Quantitative analysis of aromatase mRNA expression by real-time PCR. Relative aromatase mRNA expression was estimated using the β-tubulin mRNA expression as an internal control. (C) Promoter usage of aromatase gene expression in 5-Aza-dC-treated endometrial cells. Unique Exon I primers (PII, I.1, I.3, I.4, I.5 and I.6) and Exon II primers were used for Exon I-specific PCR. At the end of the PCR cycles, the reaction mixtures were removed and subjected to electrophoresis on a 1.5% agarose gel. Separated DNA sequences were then transferred to a nylon membrane and hybridized to 32P-labeled aromatase Exon II cDNA for 18 hours. At the end of the hybridization period, the nylon membrane was removed, washed, and exposed to x-ray film.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions