Aromatase expression in abdominal omental/visceral and subcutaneous fat depots: a comparison of pregnant and obese women Suman Rice, Ph.D., Bijal Patel,

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Aromatase expression in abdominal omental/visceral and subcutaneous fat depots: a comparison of pregnant and obese women Suman Rice, Ph.D., Bijal Patel, B.Sc., Gul Bano, M.D., F.R.C.P., Austin Ugwumadu, Ph.D., F.R.C.O.G., Saffron A. Whitehead, Ph.D. Fertility and Sterility Volume 97, Issue 6, Pages 1460-1466.e1 (June 2012) DOI: 10.1016/j.fertnstert.2012.03.008 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Column graph showing total levels of CYP19 mRNA measured in SC and OM adipose tissue samples from pregnant (n = 4) and obese women (n = 3). Aromatase mRNA was measured by real-time quantitative PCR and expressed as a fold change in expression relative to β-actin. There was a more than 10-fold increase in total aromatase mRNA in OM compared with SC in samples from pregnant women (P=.057, Mann-Whitney), and this depot-specific difference was significant using the F test for variance (∗P=.03). In samples from obese women, there was a fourfold higher increase in total aromatase mRNA in the SC compared with OM depot (∗P=.05, Mann-Whitney). Expression of aromatase was significantly up-regulated in OM tissue from pregnant women compared with obese (∗P=.03, Mann-Whitney). Fertility and Sterility 2012 97, 1460-1466.e1DOI: (10.1016/j.fertnstert.2012.03.008) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Representative picture of agarose gels (2%) showing the mRNA expression of various aromatase promoters in SC and OM adipose samples from pregnant subjects 1, 2, 5, and 6. Standard PCR analysis was performed using promoter-specific primers to investigate the difference between the two depots of mRNA expression of PII, P1.4, and P1.3. Messenger RNA from ovary (OC) and granulosa-luteal cells were included as a positive control for the promoters. Also included was a no-RT control for sample 6 (OM− and SC−), which indicated that there was no genomic contamination of RNA extracted. Beta-actin mRNA expression was used as a reference gene and was expressed in all tissue samples. Promoter 1.3 was not expressed in any adipose tissue samples. (B) Graph of densitometry values of PCR analysis of PII and P1.4 in all SC and OM samples from pregnant women (mean ± SEM). The PII expression was significantly higher in OM compared with SC (∗∗P=.004) and also higher than P1.4 in either SC (∗P=.03) or OM (∗P=.03) depots (unpaired t test with Welch's correction). Fertility and Sterility 2012 97, 1460-1466.e1DOI: (10.1016/j.fertnstert.2012.03.008) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Representative picture of agarose gels (2%) showing the mRNA expression of various aromatase promoters in SC and OM adipose samples from obese subjects 1, 2, and 3. Standard PCR analysis was performed using promoter-specific primers to investigate the difference between the two depots of mRNA expression of PII, P1.4, and P1.3. Messenger RNA from KGN was included as a positive control for the promoters. Also included was a no-RT control for sample 3 (OM−), which indicated that there was no genomic contamination of RNA extracted. Beta-actin mRNA expression was used as the reference gene and was expressed in all tissue samples. Promoter 1.3 was not expressed in any adipose tissue samples. Fertility and Sterility 2012 97, 1460-1466.e1DOI: (10.1016/j.fertnstert.2012.03.008) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Representative picture of Western blots showing the protein expression of aromatase and β-actin in SC and OM tissue samples from pregnant women (samples P1, P2, P5, and P6), with protein from placenta (plac) as a positive control from aromatase expression. There was expression of β-actin in all samples. Aromatase was detected in all samples, albeit at a much lower level than placenta, with no discernible differences between the depots. L = protein marker. (B) Representative picture of Western blots showing the protein expression of aromatase and β-actin in SC and OM tissue samples from obese women (samples Ob1, Ob2, and Ob3). There was barely any detectable aromatase protein present despite adequate amounts of β-actin. Fertility and Sterility 2012 97, 1460-1466.e1DOI: (10.1016/j.fertnstert.2012.03.008) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions