The ethics committee of Shandong Provincial Hospital has approved this experiment. Fresh placentas from full-term women were collected in the Obstetrics and Gynecology Department of Shandong Provincial Hospital. Hepatitis B and C, syphilis, HIV and fetal malformations were ruled out in the prenatal examination, and the pregnant women signed an informed consent form.

To explore whether the amniotic membrane ages at term labor, we obtained multiple samples from the rupture-zone, peri-placental zone not including the placenta and mid-zone areas from women who underwent spontaneous vaginal delivery at term (TL-AM) or elective cesarean delivery with no contraction (TNIL-AM) (McLaren et al., 1999; Nhan-Chang et al., 2010). And the senescence-associated-β-galactosidase (SA-β-gal) was performed to demonstrate cellular senescence of amniotic membrane bothTNIL-AM and TL-AM. For hAEC isolation, the amniotic membrane tissue was aseptically peeled off from the placenta, placed in sterile saline, and transported to the laboratory within 3 h. The isolation process was performed as previously described (Murphy et al., 2010). The membrane was fully rinsed with sterile saline until there was no blood attached to it. The amniotic membrane was cut into small 1 cm × 1 cm pieces, and all pieces of amniotic membrane were transferred into a sterile specimen container containing 0.05% trypsin in Hank’s solution (D-Hanks; Boster). After incubation for 60 min at 37 °C with 200 rpm shaking to digest the tissues, the digested cells were filtered with a 300-mesh cell strainer, and a same volume of DMEM (Gibco) complete medium containing 10% FBS (Gibco) was used to stop digestion. The cell suspension was centrifuged for 10 min at 1500 rpm, the supernatant was removed, and the sediment was resuspended in 1 ml of DMEM, including 10% exosome-free FBS, 1% penicillin and 100 μg/ml streptomycin (Solarbio). FBS without exosomes was obtained at 120,000×g for 18 h by ultracentrifugation (Shelke et al., 2014), and then, the supernatant was filtered through a 0.22-μm membrane (Millipore). Next, the cells were inoculated at a concentration of 1 × 10^5 into a T25 flask (Corning) and incubated at 37°C in 5% carbon dioxide (CO2) until the cell confluence reached 80–90%. The cell state was observed under a microscope (Olympus) and photographed and recorded every day.

hAECs (1 × 10^4) were seeded into 96-well plates (Corning) and incubated for 4–5 h to allow the cells to adhere to the plate walls. Then, 4% paraformaldehyde was used to fix the cells for 15 min, and cells were permeabilized with 0.5% Triton X-100 for 20 min. Lastly, goat serum (Boster) was used to block the samples for 30 min. All procedures were performed at room temperature. hAECs were identified with a rabbit monoclonal primary cytokeratin 19 antibody (Abcam) diluted 1:200 in primary antibody dilution overnight at 4°C. Next, the plates were incubated with 1:500 Alexa Fluor 488-labeled goat anti-rabbit IgG (H + L) (Beyotime) for 1 h at room temperature, and nuclei were stained with DAPI (Solarbio). Confocal microscopy was applied for imaging.

The human lung carcinoma cell lines (A549) was purchased from Bluef (Shanghai) Biotechnology Development Co., Ltd. Cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% exosome-free FBS (Gibco), 1% penicillin and streptomycin (Gibco). Cell culture was maintained at 37°C in a humidified atmosphere with 5% CO2. After 48 h of culture, the medium in each well was collected and centrifuged instantly under the condition of 800×g for 10 min to isolate cell precipitations, and then, the supernatant was stored at −80°C until analysis.

Collection of amniotic fluid (AF) samples was carried out with the support of the ethics committee of Shandong Provincial Hospital, and informed consents were obtained from patients. AF samples were collected from women who underwent 1) spontaneous term labour without complications and vaginal parturition (TL; n = 10); spontaneous term labour without complications was defined as regular, progressively increasing uterine contractions lasting 30 s or more with 5–6 min intervals, accompanied by disappearance of the cervical canal, dilation of the cervix, and descent of the fetal presentation followed by labour at ≥ 37 gestational weeks and ≤42 gestational weeks with no pregnant complications during gestation, and 2) normal term not in labour (TNIL; n = 10), defined as opting for elective cesarean operation to delivery at 37–42 weeks of normal gestation. Exclusion criteria included preterm birth, post-term pregnancy, multiple gestations, gestational hypertension/preeclampsia, placental abruption/previa, infant deformities, gestational diabetes, and intrauterine growth restriction.

For vaginal labour, AF samples were suctioned by using a 10-ml syringe through the dilated cervix before artificial rupture of membrane. For elective cesarean delivery, after exposure of the integral amniotic sac, a 10-ml syringe was used to collect AF samples to avoid contamination. Sediments in all AF samples were immediately removed by centrifuging under the condition of 2000×g for 10 min. After that, the AF supernatant was stored at −80°C in the dark until analysis.

Exosomes were isolated from AF and A549 cell supernatants as previously described (Dixon et al., 2018; Sheller-Miller and Menon, 2020). In brief, an equal volume of 1× phosphate-buffered saline (PBS) (Solarbio) was used to dilute the AF sample, which was centrifuged at 300×g for 10 min at 4°C. Then, the supernatant was centrifuged at 2,000×g for 30 min at 4°C. The resultant supernatant fluid was then centrifuged at 12,000×g for 45 min at 4°C. The sediment was discarded, and the supernatant was filtered through a 0.22-µm filter (Millipore). Next, the filtered fluid was transferred into an ultracentrifuge tube, and ultracentrifugation (Hitachi) was performed at 120,000×g for 70 min twice. The pellet containing the small vesicles was resuspended in 200 µl of 1× PBS and stored at −80°C. The above steps for AF exosome isolation are also suitable for exosomes derived from A549 cell supernatant.

C57BL/6 (B6) mice were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd., and were bred in the animal care facility at the animal laboratory of Shandong Provincial Hospital. All mice were raised under a circadian rhythm condition (dark time: light time = 12:12). Eight-week-old females, after adaptive feeding for 1 week, were mated with males of the same phenotype. From 8:00 a.m. to 9:00 a.m. on the second day, we checked daily for the appearance of a vaginal plug indicating 0.5 days post-coitum (dpc). Females were then separated from the males, and their body weight was monitored every day. Until 12.5 dpc, if a mouse gained two or more grams until 12.5 dpc, it was considered pregnant. All animal experiments has approved by the Ethics Committee of Shandong Provincial Hospital.

For assessment of exosome morphology, exosomes were resuspended in 50 µl of 1× PBS solution, and a drop of the solution was loaded onto a copper grid at room temperature for 10 min. Then, 3% glutaraldehyde was used for fixation for 5 min, and excess glutaraldehyde was blotted off with filter paper. The exosomes were then transferred to diluted water solution for 2 min, and this step was repeated 10 times. Finally, 4% uranyl acetate was used for washing. Grids were allowed to dry for approximately 30 min and subsequently imaged in a transmission electron microscope in the laboratory of Shandong Second Provincial General Hospital.

Exosomes were resuspended in 50 µl of 1× PBS, and 5 µl of resuspended solution was used for NTA. NTA measurements were performed using a ZetaView® Nanoparticle Tracking Analyzer PMX-120 following the manufacturer’s instructions. First, the instrument performance was tested, and then the samples were examined. From each sample, the particle size and concentration information can be obtained with the outer probe of the instrument.

Study has verified that extracellular vesicle (exosme) produced upon fusion of endosomal multivesicular bodies (MVBs) with the cell membrane, and CD63 is a tetraspanin which is necessary for this process. So exosome contains a higher concentration of CD63 (Mathieu et al., 2021). TSG101 is one of the main component of the endosomal sorting complex required for transport (ESCRT) being involved in MVB and exosome biogenesis, and TSG101 is detected in purified exosomes from heterogeneous cell types by proteomic analysis (Colombo et al., 2013). Alveolar epithelial type II cells (AT2) can synthetize pulmonary surfactant, and a common way to identify AT2 cells is to use SP-C which is the only AT2-specific protein (Beers and Moodley, 2017). EpCAM is a pan-epithelial cell marker, and it can be detected on the surface of AT2 (Hasegawa et al., 2017). The expression of exosomal CD63 and TSG101 and the alveolar epithelial type II cell markers EpCAM and SP-C were evaluated by western blot analysis. Exosomes isolated from amniotic fluid (AF-exos) and A549-exos were lysed using a precooled RIPA and PMSF mixed buffer (100:1) and centrifuged for 30 min at 4 °C. The exosomal protein concentration was quantified by a BCA Protein Assay Kit.

Samples were loaded on a 10% SDS–PAGE gel and then transferred onto PVDF membranes. Then, 5% skim milk dissolved in 1× TBST was used to block the membranes for 1 h at room temperature, and the membranes were probed with anti-CD63 (1:1000; Abcam), anti-TSG101 (1:1000; Abcam), anti-SP-C (1:1000; Sigma) and anti-EpCAM (1:1000; Proteintech) primary antibodies at 4°C overnight. After the membranes were washed three times with Tris-buffered saline with Tween (TBST) buffer, they were incubated with secondary horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Solarbio) at room temperature for 1 h. The blots were washed with TBST for three times, and the Amersham Imager 600 Imaging System (GE Healthcare, Chicago, IL) was used for imaging.

Fluorescently detectable AF-exos and A549-exos were generated by using PKH67 green membrane dye (Sigma). Hoechst and phalloidin were used to stain the cell nucleus and cytoskeleton, respectively. hAECs grown to approximately 80–90% confluency were washed twice with 1× PBS and cultured in a 96-well plate with medium containing 10% exosome-free FBS for 24 h. Briefly, the staining steps were as follows: 20 µl of exosomes was added to 500 µl of Diluent C (solution A) and 2 µl of PKH67 dye mixed with 500 µl of Diluent C (solution B) in the dark. Then, solutions A and B were mixed quickly and incubated for 5 min at room temperature in the dark. Next, 1 ml of 1% bovine serum albumin (BSA; Solarbio) was added to stop the labeling. The stained exosomes were resuspended in 1× PBS buffer and then ultracentrifuged at 110,000×g for 70 min at 4°C. The supernatant was eliminated, and the PKH67-labeled exosomes were resuspended in 130 μl of 1× PBS. Ten microliters of labeled exosomes was added to each well and incubated overnight at 37°C and 5% CO2. The next day, 100 µl of 4% paraformaldehyde (Solarbio) was added to each well to fix at room temperature for 15 min, followed by washing three times with 1× PBS. Then, each well received 100 µl of cell membrane permeabilization solution (Solarbio) at room temperature for 5 min. Every well was washed three times with 1× PBS, followed by the addition of 70 µl of phalloidin to each well and incubation of the plate in the dark and at room temperature for 30 min. After that, 100 µl of Hoechst was added to each well and, the samples were incubated for 5 min in a dark place. Finally, the samples were imaged using ImageXpress Micro Confocal with MetaXpress software.

For the AF-exos groups, 100 µg of TNIL-exos and 100 µg of TL-exos were dissolved in 1 ml of complete DMEM containing 5% exosome-free FBS and filtered through a 0.22-µm membrane. HAECs were cultivated in 6-well plates, and the above medium was used to culture the hAECs for 24 h.

For the A549-exos groups, 100 µg of A549-exos was added to 1 ml of DMEM complete medium including 5% exosome-free FBS as the experimental group and filtered. Complete DMEM (1 ml) containing 5% exosome-free FBS was used as the control group. The two kinds of medium were used to culture hAECs seeded in 6-well plates for 24 h.

After coincubation with TNIL-exos, TL-exos and A549-exos for 24 h, the total protein was extracted from cells, and the same amount of protein was electrophoresed on SDS-PAGE. A PVDF membrane was used to transfer the protein, followed by incubation in 5% skim milk to block for 1 h. Then, the primary antibodies anti-p38 MAPK (1:1000, Abcam), anti-pp38 MAPK (1:1000, Abcam), anti-HSP70 (1:1000, Abcam) and anti-HMGB1 (1:1000, Abcam) were added and incubated at 4°C overnight. Tubulin (1:1000, Abcam) served as the internal controls. The goat anti-rabbit and goat anti-mouse antibodies (1:1000, Solarbio) conjugated with HRP were used as secondary antibodies. Chemiluminescence detection was performed using the Amersham Imager 600 Imaging System.

All experimental reagents used for amniotic membrane staining were purchased from Servicebio company. First, the amniotic membrane was cut into slices using a freezing microtome (Thermo). Then, the working solution was prepared, the fresh slices were fixed, β-galactosidase staining was performed, the slices were dehydrated and sealed, and the senescent blue cells were observed under a microscope.

The supernatant of hAECs cocultivated with TNIL-exos, TL-exos and A549-exos for 24 h was centrifuged at 1000×g for 15 min before use and stored at −80°C. Concentrations of the SASP markers MMP9, GM-CSF, TNF-a, IL-6 and IL-8 were measured by ELISAs (Cusabio Biotech). The experimental process was performed following the manufacturer’s protocol. Briefly, all reagents, working solution, standard solution and samples were prepared as instructed, 100 μl of standard or sample was added to each well, and the plates were incubated for 2 h at 37°C. Then, 100 μl of biotin-antibody (×1) was added to each well, incubated for 1 h at 37°C, aspirated from each well and washed, and 100 μl of HRP-avidin (×1) was added to each well. The plates were incubated for 1 h at 37°C, and 90 μl of TMB substrate was added to each well and kept for 15 min at 37°C protected from light. Finally, each well was filled with 50 μl of stop solution, the plate was gently shaken to ensure thorough mixing, and a microplate reader (BioTek; United States; IL) was applied to measure the absorbance at 450 nm.

HAECs were cocultured with TNIL-exos, TL-exos and A549-exos for 24 h. According to the instruction of the apoptosis kit (BD Biosciences), an apoptosis assay was performed. Briefly, the medium was removed, and the resuspended cell solution had a concentration of 1 × 10^6 cells/ml. Then, the staining solution was mixed with 100 µl of cell suspension in 1.5-ml EP tubes for 15 min in the dark. Next, 500 µl of binding buffer was added and mixed on ice. Flow cytometry (BD Accuri™ C6 Plus) was used to detect apoptotic cells within 1 h.

Pregnant B6 mice were anesthetized at 14.5 dpc by intraperitoneal administration of 50 µl of propofol. Mice were placed on an operation table and stabilized with tape. After disinfection with iodophor, we used sterile scissors to open the abdomen of the mice and recorded the number of fetuses. A concentration of 500 µg of A549-exos dissolved in 50 µl of sterile PBS was injected into each amniotic cavity using a single-use insulin syringe (n = 12). Controls (n = 12) were treated with an equal volume of sterile 1× PBS alone (Radnaa et al., 2021). After injection with exosomes, the mice were monitored every morning (8:00 a.m.) before 19.5 dpc to determine vaginal bleeding before preterm birth (≤18.5dpc) and the rate of preterm birth. Another pregnant mouse was injected with PKH 67-labeled A549-exo at 14.5 dpc through the same way, and sacrificed by cervical dislocation after 24 h to obtain the amniotic membranes and placentas.

The intact amniotic sacs were collected by removing uterine tissue, the amniotic membranes and placentas were collected respectively. The membranes were cut into small pieces and all pieces of membranes were digested with 0.25% trypsin. After incubation for 45 min at 37°C, the digested cells were filtered with a 300-mesh cell strainer, and a same volume of DMEM/F12 complete medium containing 10% FBS was used to stop digestion. The cell suspension was centrifuged for 5 min at 1500 rpm, the supernatant was removed, and the sediment was incubated in red blood cell (RBC) lysis buffer. Then the cell suspension was centrifuged for 5 min at 1500 rpm, and the cell pellets were resuspended in 1 ml 1× PBS to remove the excess RBC lysis buffer. Lastly, the 1 ml completed medium of DMEM/F12 was used to resuspend the obtained cells followed by seeding into 96-well plates and incubating until the cells adhered to the plate walls. The placenta tissue was cut into 1 mm³ pieces and digested with collagenase Type IV at 37°C for 30 min. The following steps were the same with the process of amniotic cells isolation.

This procedure was performed to demonstrate that whether the PKH67-labeled exosomes can be taken up by amniotic cells as well as placental derived cells or not. After the isolated cells adhered to the wall, 4% paraformaldehyde was used to fix the cells for 15 min, and followed by cell nuclei staining with DAPI for 8 min. All procedures were performed at room temperature in the dark. Fluorescence microscope was applied for imaging.

Data are showed as the mean ± SD. The ImageJ software was used to analyse the western blot and immunofluorescence results, the GraphPad Prism version 7 was applied to perform the statistical analyses. The analyses of vaginal bleeding and preterm birth relied on Fisher’s exact test, and other statistical analyses were performed with Student’s t test. A p value <0.05 was defined as significant.

Under a light microscope, the TL-AM showed a higher positive staining rate than that of TNIL-AM (Figure 1A). The staining of cytokeratin 19 both TNIL-AM and TL-AM were all positive (Figure 1B). The 7-day hAECs appeared as typical pebble-shaped cells (Figure 1C). The cytokeratin 19 antibody, a marker of epithelial cells, was used to determine the purity of hAEC culture. Green fluorescence intensity could be observed, and the statistics analysis revealed a pretty high percentage of hAECs, which suggested that the about 90% isolated cell were hAECs (Figure 1J).

Exosomes were isolated as previously described, and PKH67-labeled TNIL-exos and TL-exos were taken up by hAECs (Figure 1K,L). Both TNIL-exo and TL-exo groups showed the presence of typical cup-shaped vesicles by electron microscopy (Figures 1D,E). NTA indicated that the diameter of the two kinds of exosomes were between 50 and 150 nm (Figures 1F,G). Western blotting identified exosome surface protein markers (CD63 and TSG101) and alveolar epithelial type II cell surface markers (EpCAM and SP-C, Figures 1H,I).

P38 MAPK is a member of the MAPK family and is known to play a significant role in cellular inflammation, aging and apoptosis. Therefore, activation of p38 MAPK can induce cell senescence and apoptosis, and the initiation of cell injury results in the release of DAMPs, which cause innate immune and inflammatory cascades reactions by pattern recognition receptors. In this experiment, western blot analysis revealed that the protein levels of DAMPs (HSP70 and HMGB1) were higher in the TL-exos group (Figures 2A,B). The levels of p38 MAPK and pp38 MAPK were higher than those in the TNIL-exos group (Figures 2C,D), which showed that the senescence-associated signaling pathway of hAECs were activated after treatment with TL-exos.

Flow cytometry suggested that after TL-exos intervention, hAECs had a more obvious apoptosis rate than TNIL-exos-treated hAECs, showing a pro-apoptotic effect of TL-exo (Figure 2E).

SASP markers include matrix metalloproteinases (MMPs), cytokines, growth factors, chemokines, and enzymes generating prostanoids, and they are released by senescent foetal amniotic membrane cells. In this experiment, hAECs treated with TL-exos displayed higher levels of SASPs including MMP9, TNF-α, GM-CSF, IL-6, and IL-8 than those of the control group (Figures 2F–J).

A549-exos were isolated as previously described, and electron microscopy showed the presence of representative cup-shaped vesicles with diameters ranging from 50 to 150 nm (Figure 3A). NTA showed that particles between 50 and 150 nm were obtained (Figure 3B) and positively expressed CD63, TSG101, EpCAM, and SP-C (Figure 3C). After incubation with hAECs overnight, A549-exos were taken up by hAECs (Figure 3D). Neither AF-exos nor A549-exos showed obvious differences in morphology, diameter, surface markers or the process of internalization.

The roles of the p38 MAPK pathway in senescence and apoptosis have been described above, and we also already know that DAMPs are released by injured and senescent cells. The subsequent western blot analysis showed that the protein levels of pp38 MAPK, p38 MAPK and DAMPs were higher in the A549-exos group than in the control group (Figures 4A–D). Both the AF-exos and A549-exos groups’ results revealed that exosomes from term labor amniotic fluid may induce amniotic membrane senescence.

The definition of SASPs has already been introduced, and the representative markers include MMP9, TNF-α, GM-CSF, IL-6, and IL-8. After cocultured with A549-exos, the hAEC supernatant had a higher concentration of SASPs than that of the NC group (Figures 4F–J).

After 24 h, the intact amniotic sacs and placentas of PKH67-labeled A549-exo-injected mouse were all collected (Figures 5B,C). The cell morphology of amniotic cells and placenta-derived cells was different (Figures 5D,F) and the labeled A549-exos were observed around the nucleus, which demonstrated that exosome can be taken up by amniotic membrane and placenta (Figures 5E,G). The frequency of vaginal bleeding was higher after intraamniotic injection of A549-exos than PBS [A549-exos 66.7% (8/12) versus PBS 16.7% (2/12); p = 0.0361; Figure 5H]. Apart from that, intraamniotic injection of A549-exos also showed an increased rate of preterm birth compared with that of PBS [A549-exos 83.3% (10/12) versus PBS 25% (3/12); p = 0.0123; Figure 5I].