The present study was a case-control type; participants were rigorously screened according to predetermined inclusion and exclusion criteria. A total of 60 patients who underwent early abortion from March 2024 to August 2024 in the family planning department of Shanghai First Maternity and Infant Hospital (Shanghai, China) were included in the present study. All patients were between 20 and 40 years of age, with singleton pregnancy confirmed by ultrasound at 6-10 weeks of gestational age. The patients who underwent abortion were categorized into the following three groups according to the current pregnancy: Caesarean scar pregnancy (CSP), normal intrauterine pregnancy after cesarean section (NPACS), and normal intrauterine pregnancy without a history of caesarean section (NP). A total of 20 women were included in each group. The following criteria were used for the diagnosis of CSP in the patients included in the present study: i) Absence of gestational sac in the uterine cavity and cervix on ultrasound and closure of the cervix; ii) embeddedness of the gestational sac in the uterine incisional scar; iii) thickness of uterine myometrium of 3 mm between the gestational sac and the bladder; iv) visible blood flow around the chorionic villus sac; v) visible fetal heartbeat in the gestational sac. Patients with acute inflammatory diseases of the reproductive system, chronic diseases of thyroid gland, heart, liver and kidney, history of diabetes mellitus, hyperlipidemia and smoking or drinking were excluded. Patients who met the criteria were admitted to the hospital for pre-abortion blood testing; 5 ml of blood was retained from each patient, centrifuged (2,000 x g for 10 min at room temperature), and the supernatant was frozen in a -80 refrigerator. The test was performed uniformly after the collection of blood from all patients was completed. On the day of abortion in all patients, the decidual tissue was obtained at the localization of the gestational sac, and the decidual tissue was immediately transferred to a sterile culture solution for the extraction of decidual tissue stromal cells after obtaining. All patients signed an informed consent form, and the study was approved (approval no. KS23330) by the Ethics Committee of Shanghai First Maternity and Infant Hospital (Shanghai, China) and registered with the China Clinical Trial Registry (CCTR) under the registration number: ChiCTR2400079711.

The sample size was calculated based on the results of the pre-experiment. As a result of the pre-test, the concentration of MCP-1 in the blood of women with CSP in early pregnancy was 350±150 pg/ml, that of women with NPACS was 200±150 pg/ml, and that of women with NP was 250±150 pg/ml; with α taking the value of 0.05, β taking the value of 0.1, and the degree of certainty of 1-β: 0.9. The sample size required for unilateral testing of these three groups was 18 cases each. For the three groups, a total sample size of 54 cases was required. Eventually 60 people were included in the present study.

The extraction of decidual stromal cells was performed as previously described (15). Briefly, the obtained decidual was washed in sterile phosphate-buffered saline and chopped into 1-2-mm pieces. Tissues were digested with 0.4% (g/ml) type IV collagenase (Beijing Solarbio Science & Technology Co., Ltd.) in sterile centrifuge tubes for 60 min at 37˚C in a thermostat. The cell suspension was screened for decidual stromal cells by passing through 70- and 40-µm sterile cell sieves sequentially. The filtered cell suspension was centrifuged at 134 x g for 10 min at room temperature. after discarding the supernatant, the cell pellet was resuspended in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin and 100 mg/ml streptomycin, and incubated for 2 h at 37˚C, 5% CO₂. Suspended cells such as leukocytes and erythrocytes were aspirated.

After the inclusion of 60 patients, 18 from 60 patients were selected, 6 in each group. RNA sequencing of decidual stromal cell from 6 patients was performed in each group respectively. The selection of patients for RNA sequencing was obtained using a randomization method. Computer-generated random numbers were used for patient selection from three groups. Total RNA was extracted from decidual stromal cell with the Universal RNA Extraction CZ Kit (cat. no. RNC643; ONREW; www.onrew.com) according to the manufacturer's instructions. RNA quantity was analyzed using Qubit 4.0 (Invitrogen; Thermo Fisher Scientific, Inc.) and quality examined by electrophoresis on a denaturing agarose gel. Electrophoresis was performed using a 1% agarose gel with ethidium bromide added to the gel. After electrophoresis, the RNA bands were observed by UV imaging system. RNA libraries were prepared using the VVAHTS^® Universal V8 RNA-seq Library Prep Kit for Illumina (cat. no. NR605-0, Vazyme Biotech Co., Ltd.), followed by sequencing using the Illumina NovaSeq 6000 platform (Illumina, Inc.) with the 150 paired-end sequencing strategy. Enrichment of mRNA, library construction, sequencing and data analysis were performed by Shanghai Xu Ran Biotechnology Co., Ltd. (http://www.xurangene.com). Data quality was checked by FastQC v0.11.2 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The read length was 2x150 bp. Differential gene expression was analyzed by DESeq2 (v1.16.1) (https://bioconductor.org/packages/release/bioc/html/DESeq2.html). The thresholds for determining DEGs are P<0.05 and absolute fold change ≥2.

ELISA kits were used for the detection of molecules in blood of 60 patients. Human 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) ELISA kit (cat. no. abx250429; Abbexa; www.abbexa.com), human sterol regulatory element-binding protein 2 ELISA kit (cat. no. abx250666; Abbexa), human PAR1/thrombin receptor ELISA kit (cat. no. ab283544; Abcam), human CCR2 ELISA kit (cat. no. NBP2-75114; Novus Biologicals, LLC), hydroxy-methyl-glutaryl Coenzyme A synthase (HMGCS) ELISA kit (cat. no. MBS2019446; MyBioSource, Inc.) and cholesterol assay kit (cat. no. ARG81630; Arigobio; www.arigobio.cn) were used. All operations were performed in strict accordance with the kit instructions. The kits provided the highest concentration of the standard as a positive control, and the kits buffer without antibody or samples as a negative control. A standard curve was made according to the concentration of the standard and the corresponding OD value, and the experiment was repeated three times.

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for the extraction of total RNA from decidual stromal cells, and total RNA was quantified by nanodrop spectrophotometer (Thermo Fisher Scientific, Inc.). The reverse transcription kit (Tiangen Biotech Co., Ltd.) was used to synthesize cDNA according to the manufacturer's instructions. The abundance of mRNA was evaluated by quantitative PCR using SYBR Premix Ex Taq (Takara Bio, Inc.). Initial denaturation at 95˚C for 30 sec, followed by 40 cycles of 95˚C for 5 sec (denaturation) and 60˚C for 30 sec (annealing/extension with fluorescence acquisition). The relative expression of molecular RNA was calculated using the 2^-ΔΔCq method (16) with GAPDH as an internal reference and ddH₂O was substituted for the template as a negative control. The experiment was repeated four times. The primer sequences are shown in Table I.

The SPSS version 20.0 (IBM Corp.) statistical software package was used for the statistical analysis. Data are expressed as the mean ± standard deviation (SD). T-test, paired t-test (with Welch's correction if the variance was not equal), ANOVA, chi-squared test and Fisher's exact test were used as appropriate. Two-by-two comparisons among the three groups were performed using Bonferroni-corrected ANOVA. P<0.05 was considered to indicate a statistically significant difference.

A total of 20 women with CSP, NP and NPACS were included as aforementioned. There were no significant differences in age, body mass index (BMI), number of pregnancies, weeks of gestation, or number of miscarriages among the three groups, and the number of cesarean sections between women with CSP and women with NPACS did not differ significantly. This suggested that the number of cesarean sections may not be related to CSP (Table II). A representative image of early pregnancy is demonstrated in Fig. 1.

Chemokines and chemokine receptors play important roles in both embryo implantation and tumor metastasis (7,17-19). Decidual stromal cells were obtained for chemokine and chemokine receptor assays, and the results of RNA sequencing of decidual stromal cells from three types of patients revealed that the expression of PAR-1, MCP-1 and chemokine (C-C motif) receptor 2 (CCR2) in decidual stromal cells from women with CSP was higher than that of women from the other two groups (Fig. 2A and B), Subsequently, our PCR experiments verified that the expression of PAR-1, MCP-1, and CCR2 genes in the decidual stromal cells of women with cesarean scar pregnancies was significantly higher than that of the other two groups of patients, whereas no significant differences in the expression of PAR-1, MCP-1 and CCR2 genes were observed between the two groups of NP and NPACS (Fig. 2C). Considering that there was a close relationship between MCP-1 and cholesterol (20,21), it was explored if cholesterol synthesis altered in stromal cells at the locus of the gestational sac in women with CSP. The present PCR experiments demonstrated that cholesterol synthesis of the specific transcription factor SREBP2 and the rate-limiting enzymes HMGCR and HMGCS were significantly increased in the decidual stromal cells at the localization of the gestational sacs in women with CSP, whereas no significant differences in the expression of cholesterol-synthesis-related proteins were observed between the two groups of NP and NPACS (Fig. 2D).

It was investigated whether the blood of women with CSP revealed alterations in chemokines and cholesterol synthesis. ELISA proved that the concentrations of PAR-1, MCP-1, CCR2, SREBP2, HMGCR and HMGCS in the blood of women with CSP were significantly higher than that in the two groups of NP and NPACS, and there was no significant difference in the expression of MCP-1 and cholesterol synthesis-related proteins in the blood of women between NP and NPACS (Fig. 3A). Blood tests also identified that the level of cholesterol in the blood of women with CSP was significantly higher than that of the other two groups (Fig. 3B), suggesting that the occurrence of scar pregnancies after cesarean section was associated with a high cholesterol milieu in the body.