Objective
Telomeres shorten and aggregate with cellular senescence and oxidative stress. Telomerase and its catalytic component human telomerase reverse-transcriptase regulate telomere length. The pathogenesis of preeclampsia and intrauterine growth restriction involves hypoxic stress. We aimed to assess telomere length in trophoblasts from pregnancies with those complications.
Study Design
Placental specimens from 4 groups of patients were studied: severe preeclampsia, intrauterine growth restriction, preeclampsia combined with intrauterine growth restriction, and uncomplicated (control). Telomere length and human telomerase reverse-transcriptase expression were assessed by using quantitative fluorescence-in-situ protocol and immunohistochemistry.
Results
Telomere length was significantly lower in preeclampsia, intrauterine growth restriction, and preeclampsia plus intrauterine growth restriction placentas. More aggregates were found in preeclampsia, but not in intrauterine growth restriction placentas. Human telomerase reverse-transcriptase was significantly higher in the controls compared with the other groups.
Conclusion
Telomeres are shorter in placentas from preeclampsia and intrauterine growth restriction pregnancies. Increased telomere aggregate formation in preeclampsia but not in intrauterine growth restriction pregnancies, implies different placental stress-related mechanisms in preeclampsia with or without intrauterine growth restriction.
Telomeres are nucleoprotein structures consisting of 5-15 kilo base pairs of repetitive DNA sequences, located at the termini of the chromosomes. They are essential for chromosome stability and for cell survival and protect the chromosomes from end-to-end fusion and degradation. Telomeres are progressively shortened with each cell division and also by environmental factors such as oxidative stress. Once a critical shortening of telomeres is attained, cell senescence is triggered leading to a process of tissue aging. Shortened telomeres promote cell cycle arrest, apoptosis, and genomic instability. They also tend to form aggregates, which are end-to-end fusion of telomeres, a process that is independent of telomere length. Telomere length regulation is directed by telomere-associated proteins among which is the enzyme telomerase that adds telomeric repeats to the ends of the chromosomes. Human telomerase reverse-transcriptase (hTERT) is the catalytic component of telomerase and is considered to be the rate limiting factor in telomerase activity. The absence of telomerase in normal human cells, leads to a progressive decline in telomere length, resulting in cell senescence and tissue dysfunction.
Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy-specific disorders that exemplify altered placental implantation associated with placental insufficiency. PE is manifested by maternal hypertension and proteinuria and IUGR defined as fetal growth below the 10th percentile. These complications affect both short- and long-term maternal and fetal morbidity and mortality. PE and IUGR can coincide or occur separately. Placentas from pregnancies complicated by IUGR and PE have characteristically similar structural and cellular abnormalities, although they are possibly distinguishable from each other.
Emerging data suggest that senescence may be involved in the pathogenesis of chronic human diseases and that cells undergo stress-induced premature senescence. A previous study revealed shortened telomeres and reduced levels of telomerase activity in IUGR placentas. We reported preliminary findings of increased aggregate formation in a mixed population of placentas from pregnancies complicated with PE (mild and severe) with or without IUGR; however, we did not compare those results with telomeres length.
The aim of this study was to compare telomere length, aggregate formation, and telomerase levels in trophoblasts from pregnancies complicated with PE, IUGR secondary to placental insufficiency, and both PE and IUGR, with trophoblasts from uncomplicated pregnancies. In this study, we sought to define the differences in telomere length and function among the different clinical groups with placental insufficiency.
Materials and Methods
Placental biopsies and patients selection
The study was approved by the institutional review board.
We examined placental samples from 57 third-trimester placentas. These included 4 groups of patients: 14 were derived from pregnancies complicated with PE without IUGR, 14 from pregnancies complicated with IUGR without evidence of PE, 9 from pregnancies complicated with both severe PE and IUGR, and 20 from gestational age-matched placentas from uncomplicated pregnancies that served as controls. All the placentas were taken from third-trimester pregnancies. For each study placenta, a control sample from the same gestational week was collected.
PE was defined as maternal hypertension and proteinuria (beyond 300 mg in 24 hours). The severity of PE was defined according to the standard acceptable parameters (1 or more of the following criteria: blood pressure above 160/110, proteinuria beyond 5 g in 24 hours, elevated liver enzymes, hemolysis, or platelets below 100,000 μL/mL). IUGR was defined as fetal weight below the 10 th percentile for the gestational age. We included in the study only placental specimens from pregnancies complicated with IUGR attributed to placental insufficiency after ruling out other reasons for IUGR such as intrauterine viral infections, chromosomal abnormalities congenital anomalies, poor pregnancy dating, or maternal smoking.
Histopathologic examination of the placental tissues
The placental biopsy specimens were stained with hematoxylin and eosin and evaluated for the presence of typical injuries of placental insufficiency, including maternal vascular abnormalities (maldevelopment or obstruction), inflammatory response (villitis of unknown etiology), distal villus hypoplasia, and fibrin deposition. We compared the prevalence of those histologic characteristics in placentas from the 4 study groups.
Fluorescence in situ hybridization (FISH) assay
Paraffin-embedded placental tissue sections of 4 μm were prepared for Fluorescence in situ hybridization (FISH) using Tissue Pretreatment Kit (Biomedicals, North America, Solon, OH). Slides were first placed in xylene for 10 minutes twice at room temperature, followed by fixation with acetic acid and ethanol at a ratio of 1:3 for 15 minutes. Afterward, the slides were placed in 100% ethanol for 5 minutes twice, allowed to air dry, and then warmed at 80°C for 60 minutes. Slides were then incubated for 25 minutes in a pretreatment solution (12 g of pretreatment powder in 40 mL of saline sodium citrate buffer [SSC]) at a temperature of 45°C. They were then rinsed twice in buffer SSC at room temperature, then incubated with protein digest enzyme preheated to 45°C for 45 minutes (400 μL in 40 mL of SSC), rinsed twice in buffer SSC in room temperature, dehydrated for 1 minute in ethanol 70%, 80%, 100% at room temperature, and left to air dry for 5 minutes. Afterward, the slides were placed on a 94°C warmed plate for 4 minutes.
Telomere length and number of aggregates were assessed by the fluorescent intensity of telomere FISH for each paraffin section using a CY3 labelled telomere-specific peptide nucleic acid probe (vial 2 in K 532; DAKO, Glostrup, Denmark), according to manufacturer’s instructions. The slides were counter stained with 4′-6-diamidino-w-phenylindole (DAPI) (1000 ng/mL, VYS-32-804830; Vysis) and finally overlaid with glass cover slips for observation with a fluorescent microscope. We used 1 filter for 3 colors (DAPI, TRIS, FITC), automatic exposure for both images, ×100 magnification on AX70 Olympus Provis microscope (Olympus, Tokyo, Japan). For each specimen, 100 trophoblasts were counted. The slides were blindly scored twice (3 times whenever there was a difference of more than 10% between the counts). Telomere length was quantified by signal intensity and the number of signals (telomeres). The cells were categorized as having high (strong) or low (weak) fluorescence, and by the number of telomere signals (dots per cell) <10 or ≥10. We then calculated the percentage of cells in each category for each slide.
The FISH manual counts were followed by computer-assisted analysis of digital images to ensure the accuracy of the counts. The software was developed in our laboratory (using MATLAB). It analyzes telomere intensity of staining in DAPI-stained nuclei by comparing the difference between the intensity of the brightest and the dimmest red-stained pixels in each nucleus that is stained blue, using a derivative-based algorithm. The total intensity of each telomere is calculated as the sum-of-intensity of all the pixels that belong to the telomere. The relative telomere length is determined by comparing the study samples with the controls. In addition, we compared the percentage of telomere aggregates, which appear as clusters of telomeric signals ( Figure 2 ), in the placental trophoblasts between the study and control groups.
Slides were also exposed to centromeric probes of the chromosomes 16, 18, X and Y and evaluated for aneuploidy.
Immunohistochemistry
Immunohistochemistry for hTERT was performed using Histostain-SP detection kit (Zymed Laboratories, San Francisco, CA). Sections (4 μm) from placental tissues from the 4 groups were deparaffinized in xylene and rehydrated in an ethanol gradient. For peroxidase quenching, the sections were incubated for 15 minutes in 3% hydrogen peroxide in methanol, at room temperature, and then blocked with blocking solution (reagent A from the above commercial kit) for 15 minutes and washed twice with PBS. The slides were incubated for 1 hour at room temperature with rabbit monoclonal antibody against hTERT (Novus Biologicals, Littleton, CO) at a dilution of 1:50 and then for 10 minutes with secondary antibody (Reagent B, biotinylated secondary antibody). The slides were then incubated for 10 minutes at room temperature with streptavidin and washed twice with PBS. The nuclei were stained with DAB for 3 minutes and washed with distilled water. The slides were then stained with hematoxylin and eosin, washed with water, covered, and viewed with a light microscope. The expression of hTERT was evaluated based on the intensity of the signal in the trophoblasts. We counted 100 trophoblasts per slide and defined 2 groups of cells: low intensity and high intensity. The slides were scored blindly twice.
Statistical analysis
A 2-tailed sample t test and analysis of variance as well as the nonparametric Kruskal-Wallis and Mann-Whitney U tests were applied to test the differences between the study groups, with P < .05 considered statistically significant. SPSS software (SPSS, Inc, Chicago, IL) was used for statistical analysis.
Results
The mean gestational weeks of the placentas that we studied were: 36 ± 2.43 for the IUGR placentas, 36 ± 1.41 for the PE, 36.2 ± 3.5 for the IUGR and PE, and 37.15 ± 3.9 for the control samples. No statistical differences were detected between the groups in terms of the gestational weeks.
To estimate placental insufficiency, the prevalence of the typical histopathologic injuries was evaluated in each study group. The results are summarized in the Table . A higher prevalence of placental insult was clearly noticed in the placenta specimens from the 3 study groups compared with the control group ( P < .05); however, no significant divergence between the different study groups could be shown based on histopathology alone.
