The present study was approved by the Institutional Review Board of the Pusan National University Hospital Clinical Trials Center (approval no. H-1302-005-015). Placental tissue samples were received from Pusan National University Hospital and collected after informed consent was obtained. Placenta samples were from pregnant women (Age, 31.6±3.58 years; collection dates between July 2012 and February 2014) who met the following inclusion criteria: i) Singleton pregnancy; ii) normal pregnancy at time of sample collection; and iii) healthy women with no preexisting clinical conditions, including diabetes, hypertension, or autoimmune disease. The normal placenta samples were divided into early preterm (22–29 week gestation; n=10), late preterm (30–36 week gestation; n=18), and term (37–40 week gestation; n=20) groups following labor onset. The placental tissues were previously confirmed by testing expression of tissue specific marker, corticotropin-releasing hormone (14).

BeWo human choriocarcinoma-derived cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% streptomycin/penicillin at 37°C in a humidified atmosphere of 5% CO₂, and allowed to attach during 24 h of incubation, following which the medium was removed and replaced with phenol-red free experimental medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) treated with charcoal-dextran FBS (Gibco; Thermo Fisher Scientific, Inc.) for 24 h before treatment. steroid hormones were dissolved in ethanol, diluted with experimental medium, and added to wells for 24 h at room temperature. Cells were exposed a physiological concentrations of E2 (100 nM), P4 (10 µM) or ethanol as a vehicle control (15,16).

Total RNA from cells and tissues were extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Total RNA concentration was measured using a spectrophotometer. DNA (cDNA) was prepared from total RNA (1 µg) by RT at 37°C for 60 min using M-MLV reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) and random primers (9-mers; Takara Bio, Inc., Otsu, Japan). qPCR was performed using cDNA template (2 µl) and SYBR Green (6 µl; Toyobo Life Science, Osaka, Japan) with specific primers. The primer sequences for CYP11A1, HSD3B1, CYP17A1, HSD17B3, CYP19A1 and β-actin are shown in Table I. qPCR was carried out for 40 cycles using the following parameters: denaturation at 95°C for 15 sec, annealing at 55°C for 15 sec, and extension at 72°C for 45 sec. Fluorescence intensity was measured at the end of each extension phase. The threshold value for the fluorescence intensity of all samples was set manually. The reaction cycle at which PCR products exceeded this fluorescence intensity threshold during the exponential phase of PCR amplification was considered to be the cycle of threshold (CT). Expression of the target gene was quantified relative to that of β-actin, a housekeeping gene, based on comparison of CTs at constant fluorescence intensity (17).

Protein samples were extracted from placental tissue with PRO-PREP™ solution (iNtRON Biotechnology, Seoul, Korea) and from BeWo cells with protein extraction reagent (Thermo Fisher Scientific, Inc.). A total of 25 µg of protein per lane was separated by 10–12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked for 2 h at room temperature with 5% skimmed milk or 5% donkey serum (Sigma-Aldrich; Merck KGaA) in PBS with 0.05% Tween-20 (PBS-T). Next, membranes were incubated with antibodies specific for CYP11A1 (1:1,000; cat. no. 14217, Cell Signaling Technology, Inc., Dallas, TX, USA), HSD3B1 (1:2,000; ab55268, Abcam, Cambridge, MA, USA), CYP17A1 (1:500; sc-66850, Santa Cruz Biotechnology, Inc., CA, USA), HSD17B3 (1:2,000; ab126228, Abcam), CYP19A1 (1:500; sc-14245, Santa Cruz Biotechnology, Inc.) and β-actin (1:3,000; cat. no. 4970, Cell Signaling Technology, Inc.) overnight at 4°C. Following this, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit (1:2,000; sc-2030, Santa Cruz Biotechnology, Inc.), anti-mouse (1:2,000; cat. no. 7076, Cell Signaling Technology, Inc.) and anti-goat (1:2,000; sc-2020, Santa Cruz Biotechnology, Inc.) secondary antibodies in blocking solution at room temperature for 1 h. Luminol reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to visualize antibody binding. Each blot was scanned using Gel Doc 1000, version 1.5 (Bio Rad Laboratories, Inc.), and band intensities were normalized to β-actin.

Placental tissues were fixed with 10% formalin overnight, embedded in paraffin wax, routinely processed and sectioned into 4 µm thick slices. The sections were deparaffinized with xylene, rehydrated with ethanol at graded decreasing concentrations of 100–70%, and finally washed with distilled water. The slides with placental sections were stained with hematoxylin and eosin (Sigma-Aldrich; Merck KGaA) and washed with distilled water. For immunohistochemistry (IHC), endogenous peroxidase activity was quenched using a 10 min incubation step with 3% hydrogen peroxide (H₂O₂) in methanol. Non-specific binding was blocked by incubation with 10% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) and the slides were incubated with primary antibody solution (CYP11A1 (1:200; ab175408, Abcam), HSD3B1 (1:2,000; ab55268, Abcam), CYP17A1 (1:500; sc-66850, Santa Cruz Biotechnology, Inc.) and CYP19A1 (1:500; sc-14245, Santa Cruz Biotechnology, Inc.) overnight at 4°C. The slides were then washed three times for 2 min each in PBS while slowly warming to room temperature, followed by incubation with secondary antibody (horseradish peroxidase-conjugated anti-rabbit (1:100; sc-2030, Santa Cruz Biotechnology, Inc.), anti-mouse (1:1,000; cat. no. 7076, Cell Signaling Technology, Inc.) for 10 min at room temperature. Primary antibody detection was performed with a Polink-2 Plus HRP DAB kit (GBI Labs, Bothell, WA, USA). Images of tissue were captured at ×40 using a model BX50F-3 optical microscope (Olympus, Tokyo, Japan) and examined visually.

Following E2 treatments, BeWo cell-cultured media were centrifuged at 1,300 × g for 15 min at 4°C and stored at −80°C. P4 accumulation in media was measured using a competitive enzyme immunoassay kit (582601; Cayman Chemical Company, Ann Arbor, MI, USA) following the manufacturer's protocol.

Blood was collected in plastic tubes under aseptic conditions with ethylene diamine tetra-acetic acid as an anti-coagulant, followed by centrifugation in order to separate plasma. Serum and human placenta proteins were collected from each volunteer and stored at −80°C, following which the sample was slowly thawed at room temperature. Pregnenolone (PG), dihydroepiandrosterone (DHEA), E2, P4, and testosterone (T) concentrations were measured using an ELISA kit following the manufacturer's protocol. ELISA kits for PG and DHEA were from Alpco Diagnostics (11-PREHU-E01; Salem, NH, USA) and Enzo Life Science (ADI-900-093; Ann Arbor, USA). E2, P4, and T ELISA kits were purchased from Cayman Chemical (E2, 582251; P4, 582601; T, 582701).

Results are presented as the mean ± standard deviation). Data were analyzed using a post hoc Tukey's test following one-way analysis of variance (Sigma Plot version 10.0; Systat Software, Inc., San Jose, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To evaluate the mRNA expression of steroidogenic enzymes in human placenta according to gestational age, qPCR was carried out in tissues of early preterm, late preterm and term placentas. As gestational age increased, the mRNA expression of steroidogenic enzymes was elevated in human placenta and maximized in the term period. Among the genes, CYP11A1, HSD3B1, HSD17B3 and CYP19A1 were significantly increased, compared with the early preterm group; whereas the changes in CYP17A1 expression were not significant (Fig. 1).

Protein expression levels of steroidogenesis-associated enzymes in human placenta according to gestational age were also investigated by western blotting (Fig. 2A). The protein expression of CYP11A1 and HSD3B1 was not significantly altered (Fig. 2B and C). However, similar to the mRNA expression results, the protein levels of CYP17A1, HSD17B3 and CYP19A1 were elevated in term placenta, compared with the early preterm group (Fig. 2D-F).

As steroid hormone biosynthesis-related enzymes in placenta was differentially regulated according to gestational age, the concentration of steroid hormones in human serum and placenta was determined with ELISAs. Concentrations of DHEA and P4 were higher in serum than placenta, whereas the concentration of E2 was higher in placenta than serum during all periods of gestation (Table II). PG levels were stable and similar between the serum and placenta. For gestational stage, serum levels of DHEA, P4, T and E2 were significantly elevated at term compared with the early preterm group. Concentrations of steroid hormones including DHEA and E2 in placental tissue showed similar patterns to those in serum, by elevating at term stage. Notably, the level of T in placenta was reduced in term placenta, which was opposite from serum concentration. The level of P4 was also different from serum, which was not changed according to gestational age.

To determine steroidogenic enzyme localization, immunostaining was performed in late preterm placenta. Placental tissue was stained with hematoxylin & eosin to confirm the morphology of placental tissue (Fig. 3A). Tissue sections were immunostained with specific antibodies of proteins and counter-stained with H&E to show the spatial localizations of CYP11A1 (Fig. 3B), HSD3B1 (Fig. 3C), CYP17A1 (Fig. 3D), and CYP19A1 (Fig. 3E). In the image, the cytotrophoblast section was surrounded with a layer of syncytiotrophoblasts, which is a general morphological feature of placenta. Steroidogenic enzymes, including HSD3B1, CYP17A1 and CYP19A1, were localized to both cyto- and syncytiotrophoblast cells, and signals were more dominant in syncytiotrophoblast cells. However, expression of CYP11A1 was negligible in placenta.

For the next experiment, the expression levels of steroidogenic enzymes in placental-derived BeWo cells was examined. To evaluate the mechanism of steroidogenic enzyme regulation, BeWo cells were treated with E2 and P4, which are critical hormones during pregnancy (2), for 24 h. The mRNA expression of HSD3B1, CYP17A1, and HSD17B3 was significantly elevated by E2 in BeWo cells (Fig. 4), whereas P4 did not alter mRNA expression of these steroidogenic enzymes. Similar to the mRNA results, E2 increased protein expression of steroidogenesis-associated enzymes (Fig. 5A). Translation levels of HSD3B1, CYP17A1, and HSD17B3 were elevated 2- to 3-fold in response to E2 treatment, whereas levels of CYP19A1 were not significantly altered (Fig. 5B-E).

As the expression of steroidogenic enzymes was elevated by E2, the modulation of P4 secretion was further investigate following E2 treatment in BeWo cell-cultured media. P4 expression in conditioned cultured media (control group) collected from BeWo cells was 0.27 ng/ml (Fig. 5F). E2 increased production of P4 by 3-fold compared with the control, which was consistent with the expression results of P4-metabolizing enzymes.