Study group
We prospectively identified all women with PE managed and delivered at the Department of Obstetrics, Qingdao Municipal Hospital East Campus, China, during February 1, 2021and April 30, 2021. PE was defined according to ACOG16 criteria. The control group included women with uncomplicated, normotensive singleton pregnancies who delivered healthy, appropriate-for-gestational-age babies. Exclusion criteria were stillbirth, multiple gestations, chorioamnionitis, prepregnancy hypertension, renal disease, as well as chromosomal abnormalities and fetal anatomical defects. Baseline demographic characteristics and medical history information (maternal age, gestational age, parity, maximum systolic blood pressure, maximum diastolic blood pressure) were recorded for all participants (Table 1). The study was approved by the Ethics Committee of Qingdao Municipal Hospital. Informed consent was obtained from all participants. Placental tissue and maternal blood were collected from pregnancies complicated by preeclampsia, which were classified into two groups: early-onset group (n = 10), late-onset group (n = 10) .10 women without perinatal complications who accepted elective term cesarean section were chosen as the control group.
Table 1
Comparison of general data of three groups of pregnant women
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control group(n = 15)
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early-onset group(n = 15)
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late-onset group(n = 15)
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Maternal age(mean ± SD, years)
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32.29 ± 3.41
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33.54 ± 4.89
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30.50 ± 6.31
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Gestational age at delivery(mean ± SD, wks)
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39.107 ± 0.93
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32.408 ± 2.71
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37.107 ± 1.25*
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parity
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1.57 ± 0.51
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1.46 ± 0.51
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1.50 ± 0.51
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Maximum systolic blood pressure(mean ± SD,mmHg)
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116.86 ± 7.36
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162 ± 4.02
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159.93 ± 4.41*
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Maximum diastolic blood pressure(mean ± SD,mmHg)
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70.86 ± 4.52
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96.77 ± 5.36
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96.50 ± 5.68*
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*P < 0.05 |
Tissue sampling
Patients had undergone routine blood tests on an empty stomach during routine maternity examinations. All the blood tests were performed before the preeclampsia clinical diagnosis. Blood was centrifuged and the supernatant was stored at -20°C.
Placental tissue samples were excised from randomly selected areas of central placental cotyledons within 20 min of delivery. Three fragments from each placenta were obtained and thoroughly washed in phosphate-buffered 0.9% saline (PBS) to minimize blood contamination. A 0.5 cm x 0.5 cm sample was immediately placed in formalin solution for immunohistochemical detection and the rest snap-frozen then stored at -80°C for RNA and protein analysis.
Immunohistochemistry
Immunohistochemical staining was assessed using one histological section per case (measuring 2*1 cm on average). ANX A5 expression was evaluated on formalin-fixed paraffin-embedded samples using standard immunohistochemical method. The sections were examined under a light microscope at x10 and x20 magnification (Nikon, Japan). Any staining intensity above the background of immunolabeled cells was considered positive ANXA5 expression. The mean optical density(MOD) was used to quantify the stained patches, and Image-pro plus software(Media Cybernetics, USA) was used.
RNA Extraction and cDNA Synthesis
RNA was extracted from each tissue using UNIQ-10 TRIzol® reagent (Sangon Biotech, China) according to the manufacturer's instructions. RNA concentration and purity were measured on a NanoVue ultra-trace spectrophotometer (NanoVue, China).
cDNA was synthesized with the M-MuLV RT kit (Sangon Biotech, China), following the manufacturer’s instructions, using Oligo dT as amplification primers. cDNA was stored at − 20°C until use.
Real-Time Polymerase Chain Reaction Assay
ANXA5 (Forward primer: 5′-CCCTCTCGGCTTTATGATGCTTAT-3′; Reverse primer: 5′-ATGGCTCTCAGTTCTTCAGGTGTC-3′; Amplicon size: 116 bp) mRNA expression was measured using a qPCR assay with SG Fast qPCR Master Mix®, with β-Actin (Forward primer: 5′-GGGAAATCGTGCGTGACATTAAG-3′; Reverse primer: 5′-TGTGTTGGCGTACAGGTCTTTG-3′; Amplicon size: 68 bp) as an internal control. cDNA (1 µl) was amplified in a PCR reaction (final volume 20µl) containing 2×SG Fast qPCR Master Mix®( Sangon Biotech, China) and 200 nM of each primer. PCR conditions were: initial denaturation at 95°C for 3 min; 40 cycles of amplification, comprised of denaturation at 95 ° for 3 sec, annealing at 53°C for 20 sec and elongation at 72°C for 20 sec. PCR experiments were conducted on a Stepone™ real-time PCR amplifier (USA). The relative expression of ANXA5 mRNA was calculated using the 2−ΔΔCT (Livak) method. Normalized ANXA5 transcription levels for each PE-affected or normal sample were then calculated using the following formula:
2−ΔΔCT=2−[ΔCT(test) − ΔCT(control)] = 2−([CT(ANXA5, test) − CT(β−actin, test)]−[ CT(ANXA5, control) − CT(β−actin, control)])
Western Blot Assay
Proteins were extracted with the Tissue Protein Extraction Reagent (Sangon Biotech, China), following the manufacturer’s protocol, and were stored at 4°C until use.30 µg of each protein specimen were separated by 12.5% SDS polyacrylamide gel electrophoresis and were transferred to 0.45 µm nitrocellulose membranes. The membrane was closed with 5% BSA-TBST and anti-ANX A5 rabbit polyclonal antibody (36 kDa) monoclonal antibody (Sangon Biotech, China) at a dilution of 1:500 was added overnight. Rabbit anti-β-actin protein was used as an internal reference. The membrane was washed and the goat-anti-rabbit IgG-HRP secondary antibody (dilution 1:10,000) was added. It was developed by immunoblotting chemiluminescence, exposed by X-ray method, and scanned by the imaging system, and the ratio of the grayscale of the protein blot strip to the grayscale of β-actin was used as the relative expression level of ANX A5.
Enzyme-linked immunosorbent Assay
ANX A5 protein levels in maternal blood were measured by a two-site sandwich enzyme immunoassay enzyme-linked immunosorbent assay (ELISA), with the Human Annexin A5 ELISA Kit (mlbio, China).The kit assay Human ANXA5 level in the sample, use Purified Human ANXA5 antibody to coat microtiter plate wells, make solid-phase antibody, then add ANXA5 to the wells, Combined antibody which With HRP labeled, become antibody-antigen-enzyme-antibody complex, after washing Completely, Add TMB substrate solution, TMB substrate becomes blue color At HRP enzyme-catalyzed, the reaction is terminated by the addition of a sulphuric acid solution and the color change is measured at a wavelength of 450 nm with an automated ELISA reader (Rayto RT-6100, USA). The concentration of ANXA5 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Coagulation tests Assay
The changes of coagulation function including prothrombin time(PT), activated partial thromboplastin time(APTT), fibrinogen (Fib), and thrombin time(TT) were analyzed in the peripheral blood of each group of pregnant women. The coagulation function was examined using an STA Compact Max fully automated hemagglutination and corollary reagents.
Statistical analysis
All statistical analyses were performed using SPSS 26.0 software. Measurement data were expressed as mean ± standard deviation (x ± sd). The homogeneity of variance of each group was performed using Levene's test. If the variance was uniform, one-way ANOVA was used for comparison among groups, and the LSD-t method was used for further comparison between two groups. If the variances were not consistent, the Kruskal-Wallis H test was used for inter-group comparisons, and Dunnett's T3 method was used for further comparison between the two groups; p = 0.05 was used as the test level, and p < 0.05 was regarded as statistically significant.