1. A Pilot Study to Quantitate Placental  -Catenin Using Morphometric Analysis of Tissue Microarray Sections in Women with Gestational Diabetes, HIV and Normal Healthy Pregnancies Andrew Tong 1, Albert Franco 2, Nilsa Ramirez 3,4, Thomas J Barr 3, William Beyer 3, Daniel Brazeau 1, & Patty Fan-Havard 1,4 1 School of Pharmacy & Pharmaceutical Sciences, Univ. at Buffalo; 2 College of Medicine, Univ. North Carolina; 3 Nationwide Children’s Hospital, Columbus, OH; 4 College of Medicine, OSU Background Fetal well-being is dependant on normal placental development and vascular angiogenesis for waste removal, and gases and nutrient exchange between the maternal and fetal compartments (Figure 1). β-catenin plays an important role in the regulation of vascular endothelial (VE) cell-cell adhesions and barrier function by linking the VE-cadherin junction complex (Figure 2). Gestational diabetes mellitus (GDM) is associated with a significant decrease in both placental β-catenin and VE-cadherin expression and enhanced endothelial permeability, suggesting an impaired barrier function. Treatment of HIV with protease inhibitors (PIs)-based antiretroviral therapy (ART) has been associated with insulin resistance in non-pregnant adults, which is the same mechanism for GDM. PI-based ART is the first line regimen of choice for prevention of mother-to-child HIV transmission, and the effect of PIs on placental endothelial permeability remains unexplored. Advantages of tissue microarray (TMA) technology: - limited amount of needed tissues; - conserve original tissue blocks for additional research; - experimental uniformity; and - enhanced cost efficiency (Figure 3). Figure 3. Tissue Microarray Technology Donor Microtome sectioning Recipient Figure 2.  -catenin and VE-cadherin complex Figure 1. Placental section and chorionic villi Results AgeGest. Age (wks)FW (gm)Plac. Wt (gm) Cont (n =11)26.3 + 5.338.3 + 1.53070.9*471.7 GDM (n =19)28.4 + 6.838.1 + 1.03528.4*505.5 HIV (n = 32)26.5 + 5.238.1+ 1.03030.2471.1 Methods  -catenin IRB approval was obtained from UB and OSU. Inclusion Criteria: Pregnant women >18 years old presenting as (1) HIV-positive, (2) GDM diagnosed during the present pregnancy, or (3) uncomplicated singleton pregnancies. Consecutive TMA sections containing a total of 165 placental cores from 55 women were stained with hematoxylin & eosin (H&E) and with an anti-  -catenin antibody. Aperio ScanScope CS Slide Scanner was used to capture whole-slide high-resolution images at 20X (Figure 4). Two methods were used to quantitate  -catenin expression: - Manual visual inspection by a placental pathologist using a 3-point qualitative scale (0 = negative; 1 = weakly positive; and, 2 = strongly positive); and, - Positive Pixel Count Algorithm (Aperio Technologies) with a hue setting of 0.1 and hue width of 0.5. Morphometric parameters included number of pixel counts (N) and intensity (I) for weak positive (wp), positive (p), and strong positive (sp) (Figure 5). All measurements were corrected for area counted. Mixed ordinal logistic regression model or one-way ANOVA analysis was used to compare the difference in  -catenin expression between the three groups, and a one-way student t-test was used to compare differences between women who received PI-based and non-PI based ART chemoprophylaxis. Significance set at p<0.05. Figure 4. TMA of placental sections and quantitative analysis of  -catenin expression in fetal capillaries. Hematoxylin & eosin Np = 2183 Nsp = 318 Negative = 1975 Nwp = 883 Representative outputs from core G5 (slide 1). Figure 5. Positive pixel counts Table 1. Patient demographics. Data reported as mean + S.D. *p = < 0.007 Score (SEM)p-value Cont (n = 33)1.91 + 0.30 NS GDM (n = 51)1.71 + 0.35 HIV (n = 81)1.74 + 0.47 Table 2. Mean intensity visual score # of positive pixel counts/area (mean + SEM) 3A WPp-valueP SPp-value Cont 0.52 + 0.02 (0.49 - 0.56) <0.001 3.04 + 0.06 (3.25 – 3.55) NS 2.33 + 0.09 (3.25 – 3.55) <0.0001 GDM 1.02 + 0.12 (0.81 – 1.20) 3,75 + 0.37 (3.02 – 4.48) 1.22 + 0.12 1.22 + 0.12 (0.99 – 1.45) HIV 0.71 + 0.03 (0.64 - 0.78) NS 3.67 + 0.17 (3.34 – 4.00) NS 1.59 + 0.10 1.59 + 0.10 (1.40 – 1.79) <0.001 Sum of Intensity for all pixels/area (mean + SEM) 3B WPp-valueP SP (*SEM)p-value Cont 100.4 + 3.5 (93.6 – 107.3) <0.001 450.9 + 10.1 (431.0 – 470.7) NS 178.6 + 6.3 (166.2 – 191.0) <0.0001 GDM 196.2 + 20.4 (156.1 – 236.4) 515.4 + 51.3 (414.5 – 614.4) 98.2 + 9.1 (80.3 – 116.0) HIV 136.7 + 6.7 (123.6 – 149.8) NS 496.9 + 22.9 (452.0 – 541.9) NS 127.5 + 7.7 (112.3 – 142.7) <0.001 Visual Score p-value # of positive pixel counts /area (mean + SEM) Sum of intensity /area SPp-valueSPp-value HIV Non-PI (n=17) 1.65 NS 1.31 + 0.10 (1.12 – 1.5) <0.001 105.3 + 7.6 (3.34 – 4.00) <0.001 PI (n=10) 1.90 2.09 + 0.19 1.71 – 2.48) 185.7 + 14.9 (3.25 – 3.55) Table 3A & B. Comparative morphometric parameters for  -catenin expression Table 4. Comparative  -catenin expression between women who received non-PI and PI-based regimens Preliminary Conclusions Study Objectives Initial results of  -catenin expression by visual inspection was further validated by semi-quantitative morphometric analyses. Our data suggest a significant decrease in placental  -catenin expression from HIV-infected women, unrelated to the mechanism of GDM. To determine if there is a difference in the expression of placental β-catenin in (1) HIV-positive; (2) GDM, and (3) healthy mothers using tissue microarray technology and morphometric analyses. Figure 8.