Study Design
Following approval of the Ethics Committee of the Institutional Review Board of Chang Gung Memorial Hospital (CGMH 201601484A3) and obtaining informed written consent from all subjects, this prospective study was conducted from June 2017 to January 2019.Singleton pregnant women (n = 78) were recruited to donate blood samples during the first (gestational age: 8–14 weeks), second (gestational age: 20–24 weeks), and third (gestational age: 32–38 weeks) trimesters of pregnancy, and to provide cord blood and placenta samples during delivery (Figure 1). Among the 50 participants who donated 3 serum samples, 45 women had uncomplicated pregnancies and 5 women (11%) developed preeclampsia. During the study period, 25 women with preeclampsia during the second or third trimesters were referred from other hospitals to the high-risk pregnancy prenatal care center of our institute. Thus, there were 45 women in the uncomplicated pregnancy group and 30 women in the preeclampsia group.
Women in the uncomplicated pregnancy group had no diagnoses of pre-eclampsia or hypertension during pregnancy and no hospitalization due to premature delivery or bleeding. Women in the preeclampsia group were diagnosed based on the presence of new-onset hypertension (systolic blood pressures of 140 mmHg or more, diastolic blood pressures of 90 mmHg or more, or both) on two occasions 6 h or more apart after 20 weeks of gestation, and the presence of significant proteinuria (≥300 mg/24 h). None of the patients had a previous history of any known endocrinopathy. Women were excluded if they were smokers, alcoholics, had a chronic maternal disease (essential hypertension, connective tissue diseases, hyperthyroidism, hypothyroidism, chronic glomerulonephritis, renal failure, and diabetes mellitus), or gestational diabetes.
Tissue and blood collection
Human placental tissues and blood samples of the healthy group (n = 45) and the preeclampsia group (n = 30) were collected and immediately stored at −80°C. Blood was collected in plastic tubes under aseptic conditions, with EDTA as an anti-coagulant, and centrifuged at 18,472 × g for 10 min at 4°C to separate the serum. Serum concentrations of E2 and P4 were analyzed using commercially available immunoassay systems (ADVIA Centaur XP; Siemens USA). The intra-assay and interassay coefficients of variation were 5.0% and 4.1%, respectively, for E2; and 5.2% and 3.5%, respectively, for P4.
Immunohistochemistry
Tissues were paraffin-embedded and subjected to immunohistochemical staining. For this procedure, 4 µm-sections were deparaffinized and rehydrated, rinsed in purified water, and treated with 3% H2O2 for 15 min at room temperature. After rinsing three times with purified water, they were heated in an autoclave for 1 h with 10 mM citrate buffer, and then incubated with primary antibodies against the estrogen receptor-α (ERα; EP1 diluted 1:50, Bio SB, CA, USA), ERβ (14C8 diluted 1:100, Abcam, Cambridge, UK), progesterone receptor (PR; NCL-L-PGR–312 diluted 1:50, Leica Biosystems, Benton Lane, UK), and androgen receptor (AR; Clone SP107 diluted 1:50, ZECA, CA, USA). After addition of the appropriate secondary IgG antibody, sections were incubated with DAB (K5007, Dako, Denmark) and counterstained with hematoxylin and eosin (H&E; 1.05174, Merck, MA, USA). Finally, sections were dehydrated in a graded series of ethanol, cleared with xylene, mounted using Histomount (008030, Life technologies, MD, USA), and coverslips were applied for evaluation by light microscopy.
Western blot analysis
Placental tissues were washed in PBS and lysed in RIPA Lysis Buffer (20–188, Merck, MA, USA). Proteins were separated by electrophoresis using 8% SDS/PAGE, and then transferred to PVDF Blotting Membranes (10600022, GE Healthcare, Germany). Blots were probed with a primary antibody, and then developed using ImmobilonTM Western (WBKLS0500, Millipore, MA, USA). The primary antibodies were against ERα (MA1–39540, Thermo Fisher, IL, USA), ERβ (PA1–310B, Thermo Fisher, IL, USA), PR (MA1–411, Thermo Fisher, IL, USA), AR (06–680, Millipore, CA, USA), and GAPDH (MAB374, Millipore, CA, USA).
RNA extraction and quantitative real-time reverse transcription PCR
Total RNA was isolated from placental tissues using the RNA Clean & Concentrator–5 kit (R1014, Zymo Research, CA, USA) and reverse transcribed. Quantitative real-time time reverse transcription-PCR (qRT-PCR) was performed using the Fast SYBR® Green Master Mix (Applied Biosystems, CA, USA) and the ABI 7500 Fast Real-Time PCR System (Applied Biosystems), with the primers listed in Table 1.
Statistical analysis
SPSS version 10.0 (SPSS, Inc., Chicago, IL, USA) was used for data analysis. Proportions were compared using the chi-square test or Fisher’s exact test, as appropriate, and a P value below 0.05 was considered statistically significant.