INCREASED EXPRESSION OF PENTRAXIN 3 IN PLACENTAL TISSUES FROM PATIENTS WITH UNEXPLAINED RECURRENT PREGNANCY LOSS
Zeybek S1,*, Tepeli E2, Cetin GO3, Caner V3, Senol H4, Yildirim B2, Bagci G3
*Corresponding Author: Selcan Zeybek, M.D., Department of Medical Genetics, Erzurum Regional Training and Research Hospital, Cat Volu Street, 25070, Erzurum, Turkey. Tel.: +90-506-399-2644. Fax: +90-442-232-5025. E-mail: selcankesan@yahoo.com
page: 21

MATERIALS AND METHODS

Study Subjects. In this retrospective case-control study, we evaluated placental tissues from 50 URPL patients and 50 healthy subjects who had full-term births between 2008 and 2014. Formalin-fixed/paraffin-embedded (FFPE) tissue samples that had been previously submitted for routine pathological examination were collected from the archives of the Department of Pathology, University of Pamukkale, Denizli, Turkey. The study protocol was approved by the Ethics Committee of Pamukkale University (No: 2014/1-1, Date: 01.07.2014), and all procedures were performed in conformance with the Declaration of Helsinki (2000). The inclusion criteria for study subjects were the loss of more than two pregnancies in the presence of normal conceptus and parental karyotypes. The absence of anticardiolipin antibodies, lupus anticoagulating agents, uterine anomalies (determined via ultrasonography and hysterosalpingography), hormonal imbalances (due to polycystic ovary syndrome, diabetes and untreated thyroid disease), known autoimmune disease, such as lupus erythematosus or rheumatoid arthritis, thrombophilic abnormalities (indicated by Factor V Leiden thrombophilia and prothrombin G20210A mutations), and histopathological placental anomalies, was confirmed in all included subjects. A total of 50 women met the inclusion criteria, and none had known diseases during sampling. Placental tissues were collected from healthy women with single pregnancies; no history of pregnancy loss or pregnancy complications such as preeclampsia, eclampsia, preterm birth or intrauterine growth restriction, and no histopathological placental anomalies. No control subjects had undergone a cesarean section. Because placental PTX3 expression levels were reportedly highest at fullterm pregnancy, we made comparisons of PTX3 expression levels with those in full-term placentas from control subjects [12-14]. Sample Collection. All tissue sections were reevaluated, and optimal images of maternal placental areas, including decidual cells and trophoblasts, were collected. Cells were not analyzed separately because we do not have access to a laser microdissection instrument. Two 10 μmthick slices were cut from each FFPE block with disposable blades and placed in sterile 1.5 mL centrifuge tubes for total RNA extraction. Immunohistochemical (IHC) analyses of tissues from all study subjects were performed using 4 μm-thick sections mounted on positively charged slides. RNA Extraction and cDNA Synthesis. The tissue samples were deparaffinized by twice extracting with 1 mL of xylene for 10 min., followed by rehydration through subsequent washes with 100.0, 90.0 and 70.0% ethanol diluted in RNase-free water. Total RNA was isolated from tissue samples using RNeasy® FFPE Kits (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. The concentration and purity of the total RNA samples were determined using NanoDrop 2000c (Thermo Fisher Scientific, Wilmington, MA, USA). Total RNA samples of about 2 μg were then incubated in a final reaction volume of 20 μL containing the reagents for reverse transcription using a commercial kit (High Capacity cDNA Reverse Transcription Kit; Applied Biosystems, Foster City, CA, USA). cDNAs were stored at –20 °C until use as templates in quantitative real-time polymerase chain reactions (qRT-PCRs). Quantitative Real-Time Polymerase Chain Reaction. Real-time PCR analyses were performed using the LightCycler 480 platform (Roche Diagnostics GmbH, Penzberg, Germany) with the PCR primers and Universal ProbeLibrary (UPL) probes for PTX3 and the internal reference gene (β-actin) listed in Table 1. Primer sequences for PTX3 were 5’-CGG TGC TAG AGG AGC TG-3’ and 5’-GGA ATA AAA TAG CTG TTT CAC AAC CT-3’, with UPL probe 23; and primer sequences for ACTB (β-actin) were 5’-CGA CAG GAT GCA GAA GGA G-3’ and 5’- AGG AGG AGC AAT GAT CTT GAT CT-3’, with an Universal ProbeLibrary (UPL) probe 37. Pentraxin 3 expression levels were determined in final reaction volumes of 20 μL containing 0.5 μM of each primer, 0.15 μM of probe, 4 μL of × 5 LightCycler TaqMan Master Mix, 2 μL of cDNA sample, and 13 μL of PCR-grade water. The cycle conditions were 1 cycles at 95 °C for 10 min., followed by 45 cycles at 95 °C for 10 seconds, 60 °C for 30 seconds and 72 °C for 1 second. Analyses were performed with negative control reaction mixtures containing PCR-grade water instead of DNA. Quantification of PTX3 mRNA expression levels was calculated based on the quantification cycle (Cq) for each well, and normalized to β-actin as endogenous controls in both patient and control groups. Raw data were processed using LightCycler 480 software (Roche Diagnostics GmbH, Mannheim, Germany). The expression of PTX3 mRNA was calculated using the ΔΔCq method and compared with the expression in the control group. The difference was considered significant when the p value was <0.05. The value was represented as the mean fold of RNA expression compared with the controls. β Immunohistochemical Analyses. No monoclonal antibody against PTX3 was commercially available. Thus, IHC analyses were performed using polyclonal antibody against PTX3 (dilution, 1/250; Sigma-Aldrich, St. Louis, MO, USA). Tissue sections of 4 μm in thickness were prepared from the URPL patients (n = 50) and controls (n = 50). The sections were dehydrated at 60 °C for 2 hours and were then stained using an automated staining system (Ventana Medical Systems Inc., Tuczon, AZ, USA) according to the manufacturer’s instructions. Briefly, antigen retrieval were performed by an acidic citrate buffer-based solution (CC2; Ventana Medical Systems Inc.) for 1 hour. The tissue sections were incubated with anti-PTX3 antibody for 1 hour at 37 °C. The slides were then counterstained with hematoxylin II and bluing reagent (Ventana Medical Systems Inc.) for 4 min., then dehydrated in a graded series of ethyl alcohol and xylene baths. Negative controls were achieved by substituting the primary antibody with phosphate-buffered saline, and PTX3-immunoreactive cells were counted under a light microscope with a ×40 objective lens. Cytoplasmic PTX3 staining intensities and percentages of positive cells were evaluated and recorded as 0 (no staining), 1 (1.0-10.0% staining of the cells), 2 (11.0- 25.0% staining of the cells), 3 (26.0-50.0% staining of the cells) or 4 (>50.0% staining of the cells) as described previously [15]. Statistical Analyses. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS®), version 23.0 (IBM SPSS Inc., Armonk, NY, USA). Data were expressed as mean values ± standard deviations (SDs). Differences between patient and control groups were identified using Mann-Whitney U test and were considered significant when the p value was <0.05. Pearson correlation was used to determine the correlation between the PTX3 expression levels obtained by IHC and qRT-PCR.



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