Tissue Collection

Human placental tissues from the first trimester (n = 15 total from 22, 24, 26, 27, 29, 32, 36, 40, 42, 48, and 52 dpc) were collected from clinically normal pregnancies, which were terminated voluntarily by dilation and curettage. The distribution of the number of samples according to dpc was two samples each from 32, 36, 42, and 48 dpc and one sample each from 22, 24, 26, 27, 29, 40, and 52 dpc. The samples were classified as Pregnancy Weeks 3-4 (22-29 dpc), Pregnancy Weeks 5-6 (32-40 dpc), and Pregnancy Weeks 7-8 (42-52 dpc). Informed consent was obtained from each patient before obtaining the placenta. Consent forms and protocols were approved by the Ethical Committee of Akdeniz University. Mean age of the patients was 32.5 yr (range, 28-35 yr). The tissues were placed in PBS (pH 7.4) and transported to the laboratory for separation of placental villous from other tissues, such as decidua or amnion. Tissues were fixed in Bouin fixative and embedded into paraffin for immunohis-tochemistry or were fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for ultrastructural analysis.

Immunohistochemistry

Serial sections were collected on poly-L-lysine-coated slides (Sigma, St. Louis, MO), dewaxed, dehydrated, and placed in citrate buffer. Im-munohistochemical detection procedures have been described previously. To unmask antigens, an antigen-retrieval procedure was performed by treating the samples twice in a microwave oven at 750 W for 5 min each time. After cooling for 20 min at room temperature, the sections were washed in PBS and then kept in 3% H2O2 for 15 min to remove endogenous peroxidase activity, followed by three washes with PBS. After blocking with 5% normal horse serum to reduce nonspecific binding, sections were incubated with mouse anti-CD31 (Labvision, Fremont, CA) and mouse anti-CD34 (Dako A/S, Glostrup, Denmark) primary antibodies at 4°C overnight. Thereafter, sequential 30-min incubations were performed with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) and peroxidase-labeled streptavidin. The resulting signal was developed with diaminobenzidine (Vector Laboratories) and mounted with glyc-erol-gelatin (Sigma). Negative-control staining was performed by replacing the primary antibody with the appropriate nonimmune immunoglobulin G isotype. Photomicrographs were taken with an Axioplan microscope (Zeiss, Oberkochen, Germany). canada health and care mall

TUNEL-Immunohistochemistry Double-Labeling

Apoptosis in placental tissues was detected by enzymatic labeling of DNA strand breaks using TUNEL, which was conducted as described previously. Serial paraffin sections (thickness, 5 |xm) from the placental tissues were cut and taken onto the slides covered with poly-L-lysine, and after drying, the slides were left in the incubator at 45°C overnight and at 60°C for 1 h. After deparaffinization and dehydration, slides were washed twice in PBS for 5 min. Following the incubation of slides with the permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 8 min at 4°C and washing twice with PBS for 5 min, the labeling reaction was performed using 50 |xl of TUNEL reagent for each sample except for the negative control, in which reagent without enzyme was added and incubated for 1 h at 37°C. Following PBS washing, slides were incubated with converter reagent for 30 min at 37°C. After washing, color development for localization of cells containing labeled DNA strand breaks was performed by incubating the slides with Fast Red substrate solution for 10 min. The TUNEL labeling was conducted using a Cell Death Detection kit (Roche, Mannheim, Germany) and performed according to the manufacturer’s instructions. Then, the same slides were processed for CD31 immunohistochemical staining through the same protocol described above.

Hematoxylin-Eosin Staining

Sections (thickness, 5 |xm) were cut from paraffin blocks and collected on poly-L-lysine-coated slides (Sigma). Sections were dewaxed in xylene, dehydrated in descending alcohol series, and stained by a routine hema-toxylin-eosin staining technique as described previously.

Electron Microscopy

Samples of human placental tissues were fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 4 h and postfixed in 1% phosphate-buffered osmium tetroxide for 2 h. The specimens were dehydrated in ascending concentrations of ethanol and embedded in araldite-epoxy resin. Semithin sections (thickness, 0.5 l^m) were stained with toluidine blue. Ultrathin sections (thickness, 70 nm) were double-stained with uranyl acetate and lead citrate.

Quantitative Analysis of Apoptotic Index

Quantification of apoptotic cells was accomplished by counting the cells involving apoptotic bodies in angiogenic cell cords, main vascular patterns, vasculatures with advanced lumen, and stromal cells among the branching vascular areas sighted in the microscopic field. Then, the apo-ptotic cell number was divided to total cells in the related area for determination of the apoptotic percentage. Two investigators who were blinded to the slides analyzed two sections from each sample and five areas for each of the vasculogenic stages in each section. The number of cells varied depending on the areas. However, all cells in the areas were counted, and the ratios of apoptotic cells to total cells were calculated.

Statistical Analysis

Quantitative analysis of apoptotic cell number was normally distributed as assessed by the Kolmogorov-Smirnov test. Both ANOVA and post-hoc Tukey test for pairwise comparisons were used in statistical analysis. A level of P < 0.05 was considered to be significant. Statistical calculations were performed using Sigmastat for Windows, version 2.0 (Jandel Scientific Corporation, San Rafael, CA).