The present study was conducted at Xuzhou Maternity and Child Health Care Hospital Affiliated to Xuzhou Medical University between April 2017 and December 2019. The protocol of the present study was approved by the Medical Ethics Committee of Xuzhou Maternity and Child Health Care Hospital Affiliated to Xuzhou Medical University (approval no. 201502) and all the patients included in the study provided signed informed consent. A total of 60 patients aged 23–41 years with a singleton pregnancy who were admitted to the Department of Obstetrics were recruited, including 20 cases of PPROM patients (gestational week <37 weeks), 20 cases of MPROM patients (gestational week ≥37 weeks) and 20 cases of healthy pregnancies as control (gestational week ≥37 weeks) groups. Gestational age was determined based on the first trimester ultrasound scan for all pregnancies.

The diagnosis of PROM was performed using a speculum examination by visualizing the characteristic pooling of amniotic fluid in the vagina, together with a positive test for the presence of insulin-like growth factor-binding protein (ACTIM PROM test; Oy Medix Biochemica Ab) in vaginal fluid. The exclusion criteria of all pregnancies in the present study were: Structural or chromosomal abnormalities of the fetus, signs of fetal hypoxia, signs of intrauterine fetal growth restriction, vaginal bleeding, or any medical complication such as diabetes mellitus hypertension, preeclampsia, or thyroid disease.

Placenta specimens were obtained from the chorion frondosum of the maternal surface of placenta during the cesarean section. Fetal membrane specimens were acquired near the rupture of fetal membrane. A part of the specimens was stored at −80°C for total RNA and protein extraction, the other parts were immersed into 4% paraformaldehyde at room temperature for 24 h for immunohistochemical staining.

Placenta and fetal membrane specimen sections (4 µm) were deparaffinized, rehydrated and incubated with 3% hydrogen peroxide at room temperature for 30 min for endogenous peroxidase activity blockage. Following blocking with 5% bovine serum albumin (cat. no. A3858; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature, sections were incubated with primary antibodies against human NLRP1 (cat. no. ab3683; 1:100 dilution; Abcam), human NLRRP3 (cat. no. ab214185; 1:100 dilution; Abcam), human AIM2 (cat. no. 93015; 1:100 dilution; Abcam) and human NLRC4 (cat. no. 99860; 1:100 dilution; Abcam) at 4°C overnight. Tissue sections were then rinsed with phosphate-buffered saline and treated with streptavidin-peroxidase conjugated IgG secondary antibody (cat. no. SP-9001; OriGene Technologies, Inc.) at 37°C for 30 min. The sections were then stained a brown color under a light microscope with diamino-3,3′-benzidine tetrahydrochloride (DAB; OriGene Technologies, Inc.) incubated at room temperature for 5 min. Slides were counterstained with hematoxylin at room temperature for 5 min, dehydrated and mounted. Protein levels were analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

The protein concentration of ADAMTS4 was determined using human ADAMTS4 ELISA kit (cat. no. CSB-E11848h; Cusabio Technology LLC) according to the manufacturer's instructions. The absorbance was determined using a microplate reader and all samples were tested in duplicate. The standard curves were drawn by plotting absorbance values against the gradient standard concentrations and the linear regression was then analyzed. The ADAMTS4 concentration in cell supernatants of placenta and fetal membrane was calculated according to the curve linear equation.

Placenta and fetal membranes were homogenized and the cells were lysed. In brief, ~100 mg placenta or fetal membrane tissue was homogenized at 4°C with a homogenizer. Total protein was extracted using Tissue or Cell Total Protein Extraction kit (cat. no. C510003; Sangon Biotech Co., Ltd.) according to the manufacturer's instructions. The concentration of proteins was established with the bicinchoninic acid (BCA) method with BCA Protein Concentration Assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology). Tissue homogenates were analyzed through standard immunoblotting analysis. Briefly, samples were boiled at 100°C for 5 min and 40 µg protein with equal volumes (20 µl) of each sample was loaded onto 12% sodium dodecyl sulfate-acrylamide gels. Protein was separated at a constant voltage of 120 V and transferred onto nitrocellulose filter membrane. Following blocking with 5% skimmed milk at room temperature for 2 h, membranes were incubated with antibodies against human NLRP1 (cat. no. ab3683; 1:1,000 dilution; Abcam), NLRP3 (cat. no. ab214185; 1:1,000 dilution; Abcam), AIM2 (cat. no. ab93015; 1:1,000 dilution; Abcam), NLRC4 (cat. no. ab99860; 1:1,000 dilution; Abcam), ASC (cat. no. ab155970; 1:1,000 dilution; Abcam), Casp1 (cat. no. 3866; 1:1,000 dilution; Cell Signaling Technology, Inc.), Casp1 p20 (cat. no. 4199; 1:1,000 dilution; Cell Signaling Technology, Inc.), proIL-18 (cat. no. 10663-1-AP; 1:1,000 dilution; ProteinTech Group, Inc.), IL-18 (cat. no. sc-7954; 1:200 dilution; Santa Cruz Biotechnology, Inc.), proIL-1β (cat. no. ab156791; 1:1,000 dilution; Abcam), IL-1β (cat. no. 83186; 1:1,000 dilution; Cell Signaling Technology, Inc.) and ADAMTS4 (cat. no. ab185722; 1:1,000 dilution; Abcam) at 4°C overnight. Anti-GAPDH (cat. no. ab9485; 1:1,000 dilution; Abcam) was applied as loading control. Membranes were then washed with Tris-buffered saline (TBS) containing 0.1% Tween-20 at room temperature for 5 min three times and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (cat. no. 7076; 1:10,000 dilution; Cell Signaling Technology, Inc.) or HRP-conjugated anti-rabbit IgG (cat. no. 7074; 1:10,000 dilution; Cell Signaling Technology, Inc.) at room temperature for 2 h. The blots were visualized with an enhanced chemiluminescence solution (cat. no. P0018; Beyotime Institute of Biotechnology), then exposed to film and quantified with ImageJ v2.0 software (National Institutes of Health). The expression level of target protein was calculated by dividing the intensity of the target protein by the intensity of GAPDH protein.

The frozen placenta and fetal membrane tissues were thawed and RNA was extracted using RNAiso Plus Total RNA extraction reagent (cat. no. 9109; Takara Bio, Inc.), followed by DNase I treatment. RNA concentration was analyzed using NanoDrop (NanoDrop Technologies; Thermo Fisher Scientific, Inc.) and RNA quality was quantified by the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) to produce an electrophoresis trace from which the RNA integrity number and DV200 index were calculated using the 2100 Expert Software (version no. B.02.10; Agilent Technologies, Inc.). RNA was then reverse-transcribed to cDNA on a Veriti 96-well Thermal Cycler (Applied Biosystems) with PrimeScript RT reagent kit (Takara Bio, Inc.) with thermocycling conditions of 42°C for 15 min and 85°C for 5 min. Primers were synthesized by Takara Bio, Inc. (sequences in Table I). PCR amplifications were performed on a LightCycler 480 Real-Time PCR System (Roche Diagnostics GmbH) with SYBR-Green PCR Master mix (Applied Biosystems) with thermocycling conditions of 95°C for 30 sec and 40 cycles at 95°C for 5 sec and 60°C for 30 sec. All procedures including RNA extraction, cDNA synthesis and qPCR were performed according to the manufacturer's protocols. Relative mRNA expression was calculated using the 2^−ΔΔCq method (17) following normalization to GAPDH expression.

Data are expressed as mean ± SEM and statistical analyses were performed using SPSS 23.0 software (IBM Corp.). Comparison between groups was carried out by unpaired Student's t-test, chi-square test, or one-way ANOVA followed by Bonferroni's post hoc test. Bivariate correlation analysis was performed using Pearson or Spearman rank correlation. All calculated P-values were two-sided and P<0.05 was considered to indicate a statistically significant difference.

The mRNA and protein expression of inflammasome-related receptors in placenta tissues were identified. As shown in Fig. 1A, the mRNA expression of NLRP1, NLRP3, AIM2 and NLRC4 in PPROM groups were all significantly increased compared with controls. In addition, mRNA expression of these receptors was further upregulated in MPROM groups compared with controls. As common components of several inflammasomes, the expression of ASC and Casp1 mRNA were also promoted in PPROM and MPROM groups when compared with control group. The protein levels of these inflammasome components in PPROM and MPROM groups changed in accordance with the changes in mRNA levels (Fig. 1B-D). These results suggested that the mRNA and protein expression of all inflammasome components, NLRP1, NLRP3, AIM2, NLRC4, ASC and Casp1, were upregulated in placenta tissue of PPROM patients. Notably, these trends of increasing inflammasome components expression were more pronounced in MPROM groups.

The mRNA levels of inflammasomes in fetal membrane of PPROM were notably enhanced compared with controls except for AIM2 expression (Fig. 2A). In addition to the mRNA levels, protein expression of these inflammasomes in PPROM groups was also increased compared with the control group except for the Casp1 expression (Fig. 2B-D). Consistently, mRNA and protein levels of inflammasomes in fetal membrane tissues of MPROM groups were further elevated compared with controls.

The mRNA expression of IL-18 and IL-1β in placenta and fetal membrane of PROM pregnancies was also investigated. As shown in Fig. 3A, IL-18 and IL-1β mRNA levels in PPROM groups in both placenta and fetal membrane were increased significantly compared with controls. A further increase of IL-18 and IL-1β mRNA levels in MPROM groups was observed compared with controls. Furthermore, the protein expression of proIL-18, IL-18, proIL-1β and IL-1β in both placenta and fetal membrane of PPROM patients were markedly elevated compared with controls (Fig. 3B-D). A consistent trend of an increase in protein expression of proIL-18, IL-18, proIL-1β and IL-1β was observed in MPROM group, as well as increased mRNA expression.

The expression of inflammasome components was then determined by immunohistochemical staining in vivo. As shown in Fig. 4A-C and E, NLRP1, NLRP3 and NLRC4 expression were all increased in placenta of PPROM groups compared with controls, whereas no significant difference in AIM2 expression levels was observed between PPROMs and controls (Fig. 4D). The expression of these inflammasome components were all markedly upregulated in the placenta of MPROMs compared with controls. Unexpectedly, no significant difference was observed in their expression levels in the fetal membrane of PPROMs compared with controls (Fig. 5A-E) and a promotion of inflammasome component expression was identified in the fetal membrane of MPROMs compared with controls. These results suggested that all four inflammasomes may be responsible for PROM progress.

The expression of ADAMTS4, one of the risk factors of PROM, was detected. As shown in Fig. 6A and B, ADAMTS4 protein levels in placenta and fetal membrane of PPROM and MPROM groups were all significantly increased compared with control groups. In addition to protein expression, upregulated mRNA expression of ADAMTS4 was observed in placenta and fetal membrane of PPROMs and MPROMs compared with controls (Fig. 6C). Furthermore, a general positive correlation between ADAMTS4 and all four inflammasome components in PPROMs (Fig. 6D) and MPROMs (Fig. 6E) was observed.