$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
All animal experiments were conducted in strict compliance with the ethical guidelines established by the Animal Research Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Approval No.: IACUC-20231121-13) and in accordance with the National Institutes of Health (NIH) guide for the care and use of laboratory animals (2011 edition). Furthermore, this study was performed in accordance with ARRIVE guidelines. All supplies (reagents, consumables, equipment, and software) and their corresponding manufacturer information and catalog numbers are provided in the Table of Materials to facilitate reproducibility.
1. Bacterial culture
Reference strains of Porphyromonas gingivalis and Fusobacterium nucleatum (strain identifiers provided in the Table of Materials) were handled under anaerobic conditions. Primary cultures were established on pre-reduced Columbia blood agar plates and incubated anaerobically (85% N₂, 10% H₂, and 5% CO₂) at 37 °C for 48 h. For experimental preparations, a single well-isolated colony was inoculated into 40 mL brain heart infusion broth and incubated under identical anaerobic conditions until mid-log phase growth (OD660 = 0.6–0.8). To minimize oxygen exposure, inoculation and transfer steps were performed inside an anaerobic workstation using pre-reduced media and tools. Bacterial concentrations were standardized to 1 × 108 CFU/mL for animal exposure using OD660-based quantification with a strain-specific OD–CFU standard curve generated under the same culture conditions; the calibration curve was established by serial dilution and colony counting on anaerobic agar plates with at least three independent calibrations. For trophoblast infection studies, bacterial suspensions were quantified using a validated enumeration method and diluted in antibiotic-free complete culture medium to achieve the specified multiplicity of infection (MOI) immediately before co-culture.
2. Animal housing and grouping
Specific pathogen-free C57BL/6J mice (n = 90; 60 females, 30 males) aged 6–7 weeks were housed under controlled conditions (22 °C ± 2 °C, 60% ± 10% humidity, 12 h light cycle) with ad libitum access to food and water. After 1 week of acclimatization, mice were randomly allocated into six experimental groups (n = 15/group): Normal control (NC), CAP with P. gingivalis (AP), CAP with F. nucleatum (AF), normal saline tail vein injection (VN), P. gingivalis tail vein injection (VP), and F. nucleatum tail vein injection (VF). Randomization and cage allocation were recorded to minimize allocation bias and potential cage effects.
3. Establishment of the CAP model
All surgical procedures were performed under aseptic conditions. Anesthesia was induced by intraperitoneal administration of pentobarbital sodium (1.62 mg/30 g body weight) combined with atropine sulfate (12.5 µg/30 g body weight), and adequate anesthetic depth was confirmed by the absence of pedal withdrawal reflex. Mice were positioned supine on a warming pad to maintain body temperature, and the mouth was gently opened to enable stable visualization of the bilateral maxillary first molars. Under magnification, the pulp chambers of the bilateral maxillary first molars were opened using a sterile round bur, followed by removal of coronal pulp tissue with sterile micro-instruments. Root canal orifices were identified using a small endodontic file (size 06–10), and canals were gently negotiated to achieve patency while avoiding excessive force to minimize the risk of perforation. The chamber was rinsed with sterile saline (or sterile PBS) and dried with sterile paper points prior to inoculation.
Bacterial inoculation was performed using a standardized suspension at 1 × 108 CFU/mL. To make dosing reproducible, the delivered volume was operationally fixed at 2 µL per tooth (approximately 2 × 105 CFU per tooth), delivered using a calibrated micropipette tip positioned at the chamber entrance. A sterile absorbent paper point pre-soaked with the same suspension was then placed into the pulp chamber to maintain localized exposure. Successful delivery was confirmed by visible wetting of the paper point and absence of leakage into the oral cavity. After inoculation, the chamber was air-dried until no visible liquid remained, then sealed with a light-cured dental adhesive (20 s), followed by restoration using a light-cured flowable resin composite (20 s). Seal integrity was confirmed by visual inspection and gentle probing; restorations showing gaps or early loss were re-sealed immediately. Post-procedural analgesia and monitoring were provided according to institutional animal care guidance.
Two weeks later, periapical radiographs were obtained to confirm CAP establishment. Successful establishment was defined by a clear periapical radiolucency with surrounding bone changes consistent with chronic periapical inflammation and bone destruction. Mating was initiated two weeks after infection/model establishment.
4. Establishment of tail vein injection model
Bacterial suspensions were prepared at a concentration of 1 × 108 CFU/mL in sterile saline. Mice were restrained, and the tail was warmed for 1–2 min to dilate the lateral tail veins. Injections were performed using a fine-gauge needle (typically 29–30 G) inserted bevel-up into the lateral tail vein at a shallow angle (approximately 10–15°). Correct intravenous placement was confirmed by a flash of blood in the hub and smooth infusion with low resistance; unsuccessful placement was indicated by increased resistance and/or formation of a subcutaneous bleb, in which case infusion was stopped and repeated at a more proximal site after brief re-warming. Mice received daily injections for three consecutive days (100 µL per mouse each time). After three days, females were co-housed with males, and vaginal plugs were checked the following morning. Following confirmation of vaginal plugs (GD 0.5), injections were administered every three days (100 µL per mouse each time) until gestational day 15.
5. Establishment of pregnant mouse model
Female and male mice were co-housed at a 2:1 ratio (female:male) overnight starting at 20:00 each day. The presence of a vaginal plug or a sperm-positive vaginal smear at 08:00 the next morning was designated as gestational day (GD) 0.5. For each mouse, initial body weight, prepartum weight, postpartum weight, gestational length, neonatal birth weight, litter size, and number of stillbirths were recorded. Based on previous studies29,30, delivery occurring before GD 18.5 was operationally defined as preterm birth in mice. Because gestational shortening may occur within the term range, pregnancy outcomes were interpreted with attention to the distinction between true preterm delivery and gestational shortening phenotypes.
6. Sample collection and processing
At gestational day 15, three pregnant mice per group were randomly selected for placental and vascular sampling (NC/AP/AF and VN/VP/VF). Following anesthesia induced by intraperitoneal injection of pentobarbital sodium (1.62 mg per 30 g body weight) and atropine sulfate (12.5 µg per 30 g body weight), blood samples were obtained via retro-orbital bleeding. Mice were euthanized in a carbon dioxide chamber until death was confirmed by absence of respiration, heartbeat, and corneal reflex; carbon dioxide delivery followed a gradual-fill approach consistent with institutional animal welfare guidance. Carotid artery bifurcation and placental tissues were collected promptly using sterile instruments to reduce cross-sample contamination. Placental tissue was divided into two portions: one portion was stored at −80 °C, and the other portion was fixed in 10% neutral buffered formalin for 24 h, followed by graded ethanol dehydration and paraffin embedding. The remaining mice were monitored for daily body weight, gestational length, litter size, and neonatal birth weight.
7. Cell culture and treatment
The human trophoblast cell line HTR-8/SVneo was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37 °C with 5% CO₂. For infection experiments, HTR-8/SVneo cells (1 × 106 cells) in logarithmic growth phase were seeded into 6-well plates and allowed to adhere overnight. Prior to bacterial challenge, the medium was replaced with antibiotic-free complete medium to avoid antibacterial carryover effects during co-culture. Bacterial suspensions of P. gingivalis or F. nucleatum were added at MOI = 50, and each well was adjusted to a final volume of 2.5 mL using complete culture medium. Plates were incubated for 24 h. Supernatants were collected at 24 h and clarified by brief centrifugation to remove debris prior to cytokine measurement. Adherent cells were washed twice with sterile PBS to remove non-adherent bacteria and then processed immediately for RNA or protein extraction according to the workflows described below.
8. Histological analysis of the mouse placenta
Placental tissues were fixed in 10% neutral buffered formalin for 24 h, paraffin-embedded, and sectioned at 4.5 µm thickness. Routine hematoxylin and eosin (H&E) staining was performed to examine placental morphology. For immunohistochemical staining, antigen localization was visualized using a chromogenic peroxidase substrate producing brown deposits at positive sites, followed by counterstaining with hematoxylin. Negative controls were run by replacing primary antibodies with non-immune serum.
To enhance reproducibility and transparency, immunohistochemical evaluation used a prespecified semi-quantitative scoring approach. Staining intensity was graded as 0 (none), 1 (weak), 2 (moderate), or 3 (strong), and the percentage of positive cells was scored as 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), or 4 (>75%). An immunoreactivity score was calculated by multiplying intensity and percentage scores for each field. For each placenta, at least five non-overlapping high-power fields from comparable anatomical regions were scored. Scoring was conducted independently by two observers blinded to group allocation, and disagreements were resolved by joint review.
9. Quantitative real-time PCR detection of bacterial DNA signals in the mouse carotid bifurcation and placenta
Tissue DNA was extracted using a silica column-based tissue DNA extraction workflow. Tissue lysates were clarified by centrifugation at 12,000 × g for 5 min at 20–25 °C, and supernatants were applied to silica columns for DNA binding by centrifugation at 12,000 × g for 1 min at 20–25 °C. Wash steps were performed according to the workflow, with each wash followed by centrifugation at 12,000 × g for 1 min at 20–25 °C. A final dry spin was performed at 12,000 × g for 2 min at 20–25 °C to remove residual ethanol. DNA was eluted in 50 µL elution buffer after a 2-min incubation at 20–25 °C and collected by centrifugation at 12,000 × g for 1 min at 20–25 °C. Bacterial genomic DNA was extracted using a silica column-based bacterial DNA extraction workflow using the same binding/wash/dry spin/elution centrifugation parameters unless otherwise required by the workflow.
DNA concentration and purity were measured using a microvolume nucleic acid quantifier, and integrity was assessed by agarose gel electrophoresis. Qualified DNA samples were stored at –20 °C for subsequent analysis. Target bacterial genes were amplified and cloned using primers listed in Supplementary Table 1, with PCR conditions detailed in Supplementary Table 2 and Supplementary Table 3. Amplified target bands were purified, cloned according to Supplementary Table 4 specifications, and transformed into competent Escherichia coli DH5α cells. Recombinant plasmid-containing strains were verified to ensure correct insert sequences, and plasmid DNA was extracted for standard preparation. Standard curves were generated by performing qPCR on serially diluted plasmid standards containing target bacterial gene fragments. The P. gingivalis 16S and F. nucleatum 16S bacterial DNA signals in carotid bifurcation and placental samples were quantitatively detected by real-time PCR, with DNA copy numbers calculated from Ct values using the standard curve. Reaction systems and programs are detailed in Supplementary Table 5 and Supplementary Table 6, respectively. Because qPCR indicates bacterial DNA signals rather than viability or cellular localization, interpretations regarding microbial presence at the maternal-fetal interface remain cautious.
10. ELISA detection of inflammatory factors in mouse serum samples and cell culture supernatants
All reagents and samples were equilibrated to room temperature prior to testing. Serum levels of TNF-α and IL-1β were measured using mouse ELISA workflows, and TNF-α and IL-1β levels in cell culture supernatants were determined using human ELISA workflows, following the kit procedures. Standards and samples were measured in duplicate wells, and standard curves were generated for each plate. Technical duplicates were averaged to yield one value per biological replicate.
11. Other molecular biology analyses
For qRT-PCR, total RNA was extracted from cultured cells using a phenol-chloroform-based RNA extraction workflow. After phase separation, the aqueous phase was clarified by centrifugation at 12,000 × g for 10 min at 4 °C, and RNA was precipitated, washed, and resuspended according to the workflow. RNA concentration was measured using a nucleic acid analyzer, and RNA integrity was assessed by agarose gel electrophoresis. Reverse transcription was performed to generate cDNA, and quantitative real-time PCR was used to quantify TNF-α and IL-1β mRNA levels. Primer sequences are listed in Supplementary Table 7, reaction mixtures in Supplementary Table 8, and reaction programs in Supplementary Table 9.
For Western blotting, protein lysates from mouse placental tissues or trophoblast cells were clarified by centrifugation at 12,000 × g for 15 min at 4 °C, quantified, and subjected to SDS-PAGE followed by membrane transfer and antibody-based detection using a fluorescence imaging workflow. Equal protein loading was set at 30 µg per lane. Original, uncropped membrane images, including molecular weight markers, were retained for all blots presented in the figures and are available DOI: 10.57760/sciencedb.28254. Unless otherwise specified, “n” refers to biological replicates (individual animals or independent cell cultures), while technical repeats are used for quality control and do not replace biological replication.
12. Statistical analysis
Statistical analyses were performed using statistical analysis software, and figures were generated using graphing software (listed in the Table of Materials). Normally distributed continuous data were presented as mean ± standard deviation and analyzed by one-way ANOVA with appropriate post hoc tests, while non-normally distributed data were expressed as median (interquartile range) and analyzed using the Kruskal–Wallis test with appropriate pairwise comparisons. Statistical significance thresholds were set at P < 0.05, P < 0.01, and P < 0.001. Unless otherwise specified, “n” denotes biological replicates, and technical replicates (e.g., duplicate ELISA wells) were averaged to yield a single value per biological replicate.