This was a prospective cohort study that included 15 pregnant women who developed LOPE after reaching a gestational age of 34 weeks or more, and 18 healthy pregnant women who delivered at full term (37 weeks or more), conducted between 2012 and 2018, at the First Affiliated Hospital of Hainan Medical College. All participants were carrying single pregnancies, and placental samples were obtained through elective cesarean section. Ethical approval for the study was granted by the Ethics Committee of the First Affiliated Hospital of Hainan Medical College. All participants provided written informed consent.

After performing a cesarean section, placental tissues were collected and subsequently incubated in 10% formalin for 48 hours. Next, the tissues underwent a dehydration process using alcohol, and diaphanization using xylene before being embedded in paraffin.

The placental tissues were permeabilized for 5 minutes with phosphate buffered saline (PBS) containing 0.2% Triton X-100 (#X100, Sigma, St. Louis, MO, USA) and 10% goat serum (#50197Z, Thermo Fisher Scientific. Waltham, MA, USA). Subsequently, they were incubated overnight at 4 °C with an anti-FOXA1 antibody (Abcam, Cambridge, MA, USA) or an anti-cytokeratin7 (CK7) antibody (Abcam, Cambridge, MA, USA). Following PBS washes, the sections were incubated with a fluorescein isothiocyanate (FITC)-conjugated or Cy5-conjugated secondary antibody (Abcam, Cambridge, MA, USA) for 1 hour at 37 °C. After additional PBS washes, the nuclei were counterstained with 4${}^{\prime }$,6-diamidino-2-phenylindole (DAPI, #D9542, Sigma, St. Louis, MO, USA). The expression of the target proteins was observed using a confocal laser-scanning microscope (Olympus, Tokyo, Japan).

The IHC analysis of FOXA1 was conducted following the methods described in a previous study [22]. The IHC staining score for placental trophoblasts was determined based on the intensity of staining and the percentage of positive cells. Depending on the staining intensity, it was categorized as follows: “intense staining” (dark brown), “moderate staining” (brown yellow), “weak staining” (light yellow), or “non-staining” (no staining), corresponding to scores of 3, 2, 1, and 0 points, respectively. Additionally, based on the percentage of positive cells, it was divided into the following groups: 0, 10%, 10%–50%, 50%–80%, and $>$80%, with corresponding scores of 0, 1, 2, 3, and 4 points. The final score was calculated using the formula: “number of positive cells score” $\times$ “staining degree score”. A score of “$>$8 points” indicated low expression, while a score of “$\ge$8 points” indicated high expression.

Human HTR-8/SVneo cells were obtained from Shanghai Institute of Cell Biology (Shanghai, China) in 2017. The cell lines were verified by CK7 immunofluorescent staining, and have been tested for mycoplasma contamination and the test outcome is negative. The cell lines were cultured in HERA CELL 150 medium (Thermo, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (#10099141; Thermo Fisher Scientific). The cells were maintained in a humidified incubator (HERA CELL 150; Thermo Fisher Scientific) with 5% CO${}_2$ at 37 °C. The green fluorescent protein (GFP) plasmid (pSIN-EF2-Pur-FLAG-ENNSPC syl) was obtained from IGE Biotech (Guangzhou, Guangdong, China), while the FOXA1 plasmid was constructed and verified through restrictive enzyme analysis and DNA sequencing (the detailed map of the FOXA1-overexpressing plasmid is shown in Supplementary Fig. 1). The DNA sequencing confirmed the successful construction of the FOXA1 plasmid. When the HTR-8/SVneo cells reached 40–50% confluence, they were infected with retrovirus-carrying the FOXA1 plasmid. After 48 hours of infection, cells were collected for further assays.

Total RNA from 1 $\times$ 10${}^6$ cells was prepared by TRIzol extraction (#15596026; Invitrogen, Carlsbad, CA, USA). 1 µg mRNA was reversely transcribed into cDNA, and the reverse transcription reaction was performed using 15 mins at 42 °C and 5 sec at 85 °C. Transcripts were quantified by qPCR on the Mx3000P qPCR system (Agilent Technologies, Santa Clara, USA). The cycling conditions were as follows: 95 °C for 30 sec, 40 cycles of 95 °C for 5 sec and 60 °C for 30 sec, followed by 95 °C for 15 sec, 60 °C for 60 sec and 95 °C for 15 sec. The forward and reverse PCR primers (5${}^{\prime }$-3${}^{\prime }$) were as follows: FOXA1 (human): forward (F): 5${}^{\prime }$-GGAAGGGCATGAAACCAGC-3${}^{\prime }$, reverse (R): 5${}^{\prime }$-CGTTGGCATAGGACATGT-3${}^{\prime }$; N-cadherin (human): F: 5${}^{\prime }$-GCCCAAGACAAAGAGACCCA-3${}^{\prime }$, R: 5${}^{\prime }$-ACCCAGTCTCTTCTGCCT-3${}^{\prime }$; vimetin (human): F: 5${}^{\prime }$-AGCTCTTGACCTTGAACGCA-3${}^{\prime }$, R: 5${}^{\prime }$-CGTGAGGTCAGGCTTGGAAA3${}^{\prime }$; fibronectin (human): F: 5${}^{\prime }$-AAGAGGCAGGCTCAGCAAAT-3${}^{\prime }$, R: 5${}^{\prime }$-TCGCAGTTAAAACCTCGGCT-3${}^{\prime }$; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (human): F: 5${}^{\prime }$-CGA-GAT-CCCTCCAAAATCAA-3${}^{\prime }$, R: 5${}^{\prime }$-TGTGGTCATGAGTCCTTCCA-3${}^{\prime }$. For qPCR normalization, GAPDH was used as the internal control, and 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method was used for calculating the relative mRNA expression.

Protein extraction from HTR-8/SVneo cells (1 $\times$ 10${}^6$) infected with the retrovirus carrying the plasmid was performed using radio immunoprecipitation assay (RIPA) buffer (#89901; Thermo Fisher Scientific, Waltham, MA, USA). A total of 30 µg of proteins were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to nitrocellulose membranes (#88025; Thermo Fisher Scientific, Waltham, MA, USA) at 300 mA for 1 hour. These membranes were subsequently blocked with 5% bovine serum albumin (BSA; #A8531, Sigma, St. Louis, MO, USA) in Tris buffered saline with Tween 20 for 1 hour at room temperature. Following this, they were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-FOXA1 (diluted at 1:1000, Abcam, Cambridge, MA, USA) and mouse monoclonal anti-human GAPDH (diluted at 1:10,000, Abcam, Cambridge, MA, USA) antibodies. Afterward, the membranes were incubated with secondary antibodies for 1 hour at room temperature. Finally, the membranes were visualized using the ECL kit (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as the reference for normalizing the expression of the target protein.

Cell apoptosis was assessed using the FITC-Annexin V/propidium iodide (PI) Apoptosis Detection kit (Thermo Fisher Scientific, Waltham, MA, USA). The transfected cells were collected, ensuring that suspension cells were also included, and washed twice with cold PBS. Subsequently, the cells were resuspended in 1$\times$ Binding Buffer and then incubated with FITC -Annexin V/PI in the dark for 15 minutes. A flow cytometer (BD Bioscience, Oxford, UK) was used to analyze the rates of cell apoptosis. For cell cycle analysis, the PI Cell Cycle analysis kit (BD Biosciences, Franklin Lakes, NJ, USA) was employed.

The Transwell invasion assay was used to assess cell invasion. In brief, the transfected cells were seeded onto the top chamber of a Transwell (8 µm) that was coated with Matrigel (Corning, New York, NY, USA). The upper chamber was filled with Roswell Park Memorial Institute (RPMI) 1640 medium (#11875119; Thermo Fisher Scientific, Waltham, MA, USA) without FBS, while the lower chamber contained RPMI 1640 medium supplemented with 10% FBS (#10099141; Thermo Fisher Scientific, Waltham, MA, USA). After an additional 24 hours of incubation, the invaded cells were fixed using 4% paraformaldehyde (#158127; Sigma, St. Louis, MO, USA) for 30 minutes and then stained with Giemsa (#G4507; Sigma, St. Louis, MO, USA). Cells that had invaded the lower membrane surface were counted in ten random fields (10$\times$ magnification).

Cell migration was evaluated using a wound healing assay [21]. Briefly, transfected HTR-8/SVneo cells were seeded in six-well plates (#140675; Thermo Fisher Scientific, Waltham, MA, USA) and cultured for 24 hours. Subsequently, an artificial wound was created using a 200 µL pipette tip. The width of the wound was measured by ImageJ software at 0, 6, and 9 hours, respectively.

For all statistical analyses, SPSS 22.0 software (IBM Corp., Armonk, NY, USA) was utilized. The normal distribution of the data were assessed by Kolmogorov-Smirnov test. Student’s t-test was employed to compare measurement data of normal distribution between two groups. The data were presented as means $\pm$ standard deviation (SD). p 0.05 was considered statistically significant.

To determine the FOXA1 expression in placental tissues of patients with LOPE, we performed immunofluorescence double-staining of placental tissue, captured under a laser confocal fluorescence microscope (Fig. 1). In the image from the normal group, the placental villi appear well-matured, with a relatively uniform matrix and a plump shape. Endothelial cells are intact, and the nuclei between syncytiotrophoblasts are neatly arranged. Additionally, a few red blood cells can be observed within the villi capillary lumens. In contrast, the LOPE group exhibits disordered nuclei in the trophoblasts, along with vascular reduction, lumen stenosis, and incomplete endothelial cells. The immunofluorescent staining reveals a weaker fluorescence intensity of FOXA1 in LOPE placental tissue compared to the normal group. CK7, a trophoblast-specific marker protein, is expressed in both cytotrophoblast and syncytiotrophoblast cells. Under low magnification, the study reveals FOXA1 expression in both syncytiotrophoblast and cytotrophoblast cells within the villi. When observed at 60$\times$ magnification, FOXA1 expression is prominently observed in the nuclei and cytoplasm of trophoblast cells.

Fig. 1.

Immunofluorescent staining of the FOXA1 in the placental tissues from normal pregnant women and women with LOPE. DAPI, diamidino-2-phenylindole; FOXA1, Forkhead Box A1; LOPE, late-onset preeclampsia; CK7, cytokeratin7.

Next, in order to confirm if FOXA1 expression was altered in LOPE placental tissues, we performed IHC staining analysis of FOXA1 in placental tissues (Fig. 2). The findings reveal a significantly reduced FOXA1 protein expression in the LOPE group when compared to the normal group.

Fig. 2.

Immunohistochemical (IHC) staining of FOXA1. (A) IHC staining of FOXA1 in the placental tissues from normal pregnant women and women with LOPE. (B) IHC scores of FOXA1 expression in the placental tissues were compared between the normal group and LOPE group. Bar results represent the mean $\pm$ standard deviation (SD). Student’s t-test, *p 0.05.

No notable differences were detected in delivery times, pregnancy times, maternal age, 1- and 5-minute Apgar scores between the normal and LOPE group. However, the gestation week at delivery was notably shorter in LOPE group compared to normal group. Patients in the LOPE group exhibited higher systolic and diastolic blood pressure in comparison to the normal group. Furthermore, 24-hour proteinuria was detected in the LOPE group, but not in the normal group. Additionally, both placental weight and neonate birth weight were lower in the LOPE group, compared to the normal group (Table 1).

To create trophoblasts overexpressing FOXA1, we introduced a retrovirus designed to overexpress FOXA1 into HTR-8/SVneo cells. This resulted in a significant elevation in FOXA1 expression levels compared to the negative control (NC) group (Fig. 3A,B), determined by qPCR and confirmed by western blot assay. Subsequently, we assessed cell apoptosis/cycle through flow cytometry. FOXA1 did not exert any significant effect on the cell apoptotic rate of trophoblasts when compared to the NC group (Fig. 4A). Furthermore, FOXA1 overexpression significantly increased S phase cell population while decreasing the G2/M phase population, without affecting G0/G1 phase population, compared to NC group (Fig. 4B).

Fig. 3.

Overexpression of FOXA1 in the trophoblast cells. (A) The mRNA expression of FOXA1 in the HTR-8/SVneo cells from NC and FOXA1 group was determined by real-time quantitative polymerase chain reaction (qPCR). (B) The expression of FOXA1 protein in the HTR-8/SVneo cells from NC and FOXA1 group was determined by western blot assay. FOXA1, Forkhead Box A1; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bar results represent the mean $\pm$ SD. n = 3. Student’s t-test, ***p 0.001.

Fig. 4.

Effects of FOXA1 overexpression on the cell apoptosis and cell cycle of trophoblast cells. (A) The cell apoptosis of HTR-8/SVneo cells from NC and FOXA1 group was determined by flow cytometry. (B) The cell cycle of HTR-8/SVneo cells from NC and FOXA1 group was determined by flow cytometry. FOXA1, Forkhead Box A1; NC, negative control. Bar results represent the mean $\pm$ SD. n = 3. Student’s t-test, *p 0.05 and ***p 0.001; ns, not significant.

To further evaluate migration/invasion of trophoblasts, transwell invasion assay deciphered that FOXA1 overexpression enhanced invasive capacity of trophoblasts compared to NC group (Fig. 5). Wound healing assay revealed that FOXA1 overexpression significantly accelerated the closure of wounds in trophoblasts when compared to NC group (Fig. 6).

Fig. 5.

Effects of FOXA1 overexpression on the cell invasion of trophoblast cells. The cell invasion of HTR-8/SVneo cells from NC and FOXA1 group was determined by Transwell invasion assay. FOXA1, Forkhead Box A1; NC, negative control. Bar results represent the mean $\pm$ SD. n = 3. Student’s t-test, **p 0.01.

Fig. 6.

Effects of FOXA1 overexpression on the cell migration of trophoblast cells. The cell migration of HTR-8/SVneo cells from NC and FOXA1 group was determined by wound healing assay. Scale bar = 100 µm. FOXA1, Forkhead Box A1; NC, negative control. Bar results represent the mean $\pm$ SD. n = 3. Student’s t-test, *p 0.05.

To further determine the mechanism responsible for FOXA1-mediated invasion and migration in trophoblast cells, qPCR was performed to determine the mRNA expression levels of N-cadherin, vimentin and fibronectin. As shown in Fig. 7, FOXA1 had no effect on the mRNA expression levels of N-cadherin, vimentin or fibronectin comparing to the NC group.

Fig. 7.

Effects of FOXA1 overexpression on the mRNA expression of N-cadherin, vimentin and fibronectin in trophoblast cells. The mRNA expressin levels of N-cadherin, vimentin and fibronectin in HTR-8/SVneo cells from NC and FOXA1 group was determined by qPCR. GAPDH was used as the internal control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FOXA1, Forkhead Box A1; NC, negative control. Bar results represent the mean $\pm$ SD. n = 3. Student’s t-test.