Abstract
Background
Maternal air pollutants exposure is associated with a number of adverse pregnancy outcomes, including recurrent spontaneous abortion (RSA). However, the underlying mechanisms are still unknown. The present study aimed to understand the mechanism of RSA and its relationship with air pollution exposure. We compared data of decidual tissue from individuals with induced abortions and those with RSA by bulk RNA sequencing (RNA-seq), reduced representation bisulfite sequencing (RRBS), and single-cell RNA sequencing (scRNA-seq). Differentially expressed genes (DEGs) were verified using RT-qPCR and pyrosequencing. A logistic regression model was used to investigate the association between air pollutants exposure and RSA.
Results
We identified 98 DEGs with aberrant methylation by overlap** the RRBS and RNA-seq data. Nineteen immune cell subsets were identified. Compared with normal controls, NK cells and macrophages accounted for different proportions in the decidua of patients with RSA. We observed that the methylation and expression of IGF2BP1 were different between patients with RSA and controls. Furthermore, we observed significant positive associations between maternal air pollutants exposure during the year prior to pregnancy and in early pregnancy and the risk of RSA. Mediation analyses suggested that 24.5% of the effects of air pollution on the risk of RSA were mediated through IGF2BP1 methylation.
Conclusion
These findings reveal a comprehensive cellular and molecular mechanism of RSA and suggest that air pollution might cause pregnancy loss by affecting the methylation level of the IGF2BP1 promoter.
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Background
Ambient air pollution has become a global environmental threat [1]. The primary air pollutants mainly include particulate matter (PM), nitrogen dioxide (NO2), carbon monoxide (CO), sulfur dioxide (SO2), and ozone (O3), among which PM remains one of the most harmful forms, contributing to more than 4.2 million premature mortalities [2]. Evidence from animal and human studies suggests that exposure to air pollution can reduce fertility rates and increase the risk of miscarriage [3]. When the concentration of ambient PM is more than 40 µg/m3, an estimated 3.5 million pregnant women experience a miscarriage per year in South Asia [4]. Besides, exposure to NO2 during early pregnancy was associated with increased odds of spontaneous abortion in linear dose–response manners [5]. A case–crossover analysis reported that PM10 (particles < 10 μm), PM2.5–10 (particles between 2.5 μm and 10 μm), and PM2.5 exposure were positively associated with an increased risk of spontaneous abortion in the USA [6]. CO exposure during the first trimester of pregnancy was linked to an increased risk of spontaneous abortion in case–control research from Iran, which reported a 95 percent increase in spontaneous abortions in cases compared to controls [7]. In some cities of China such as Jiangsu, Bei**g, and Wuhan, the researchers also found that maternal exposure to air pollution was significantly associated with an increased risk of incident spontaneous pregnancy loss [8,9,10]. Although ambient air pollution has been linked with spontaneous abortion [11, 12], the relationship between recurrent spontaneous abortion (RSA) and air pollution and its underlying molecular mechanism is not well established.
RSA, one of the most common complications of pregnancy, is usually defined as three or more consecutive spontaneous abortions with the same spouse [13]. Its incidence is 2–4% among clinically recognized pregnancies [14]. RSA is a multifactorial disease condition, which has been associated with environmental pollution, chromosomal abnormalities, genital tract anatomic abnormalities, endocrine disorders, autoimmunological factors, and infectious diseases. To date, several foundational studies have employed single-cell RNA sequencing (scRNA-seq) technology to investigate the cellular composition and inter-cellular communication events at the maternal–fetal interface in patients with RSA [15,16,17]. Advancements in scRNA-seq have greatly facilitated the development of novel approaches to improve targeted therapies and precision medicines [18]. However, these studies only analyzed the pathological mechanism of RSA from the perspective of immunology and did not completely construct all cell atlas of RSA patients. A more comprehensive cell atlas is therefore needed and provides new insights and bases for studying the pathogenesis and the clinical treatment of RSA.
Accumulating evidence indicates that exposure to air pollution, especially PM2.5, causes changes in DNA methylation both in vivo and in vitro models [19, 20]. Air pollution particles have been shown to translocate into and cross the placenta, which may occur by altering placental DNA methylation patterns that lead to changes in placental function and morphology [21, 22]. DNA methylation, one of the major epigenetic modifications, plays a vital role in the regulation of gene expression, genome stabilization, X-inactivation, genomic imprinting, and chromatin modification [23]. The aberrant methylation in the placenta is more likely to contribute to the onset of diseases such as RSA, preeclampsia, gestational diabetes, and preterm birth (PTB) [24,70]. The average air pollutant exposures were calculated for each participant for each month of the 1 year prior to conception (from 1 month prior to pregnancy to 12 months prior to pregnancy) and early pregnancy was used for subsequent analysis. A logistic regression model adjusting for the age of the mother was used to estimate the odds ratio (OR) and 95% confidence intervals (CI) for increased pregnancy loss with exposure to air pollutants of 77 participants enrolled. We also performed a stratified analysis to investigate the potential health effects modified by season (warm: April to September; cool: October to March). In our study, we assigned 43 subjects including 11 patients with RSA and 32 controls to the warm season and 34 subjects including 20 patients with RSA and 14 controls to the cool season based on the date of surgery. The correlations between methylation levels of IGF2BP1 and air pollutants prior to 1 year to pregnancy were assessed using Spearman correlation coefficients. Methylation levels of IGF2BP1 at differentially methylated CpG dinucleotide (site4, site7, and site9) were ln-transformed to approximate normal distributions. Additionally, we constructed a linear regression model on each CpG dinucleotide of IGF2BP1 adjusting for the age of the mother in 77 participants to evaluate the associations between methylation and air pollution. PLS-PM was carried out to evaluate the potential mediating effect of the methylation level of the IGF2BP1 promoter on the association between air pollution and RSA in 77 participants using SmartPLS (version 3.3.3). In PLS-PM, latent variables are used to consider all relationships between explanatory and manifest variables [71]. To test the overall model fit for PLS-SEM, GoF was calculated to identify the PLS model globally [72]. The Fisher’s exact test, Spearman correlation analysis, t test, Mann–Whitney U test, and chi-square test were performed using the SPSS software (Version 25.0).
Data availability
Datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
Abbreviations
- DEGs:
-
Differentially expressed genes
- DMG:
-
Differentially methylated gene
- DMRs:
-
Differentially methylated regions
- m6A:
-
N6-methyladenosine
- PLS-PM:
-
Partial least squares path modeling
- PM:
-
Particulate matter
- PTB:
-
Preterm birth
- RSA:
-
Recurrent spontaneous abortion
- RRBS:
-
Reduced representation bisulfite sequencing
- scRNA-seq:
-
Single-cell RNA sequencing
- TF:
-
Transcription factor
- NO2 :
-
Nitrogen oxide
- CO:
-
Carbon monoxide
- SO2 :
-
Sulfur dioxide
- O3 :
-
Ozone
- GoF:
-
Goodness of fit (GoF)
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Acknowledgements
We would like to thank Yawei **n for assisting in sample collection and collation. We also thank all participants who were included in the study for their valuable contribution.
Funding
This work was supported by Grants from the National Natural Science Foundation of China (Nos. 82171655, 81771655, 81571503, and 81701445) and Natural Science Foundation of Shanghai (No. 22ZR1456200). The authors are grateful to all the members for their generous participation.
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WZ was responsible for data curation and original draft preparation. YG, ZZ, and JL were responsible for resources and ample collection. ML, YM, QZ, and JD were responsible for methodology and review and editing. LZ, YS, FC, JX, and LH were responsible for article review and editing. All authors read and approved the final manuscript.
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All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Shanghai Institute of Planned Parenthood Research.
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Additional file 1
: Table 1: Primers for genes validated using qRT-PCR and pyrosequencing sequencing primers. Table 3. The methylation levels of CpG sites in the promoter regions of FLT1 and IGF2BP1 were detected by pyrosequencing. Table 4: Descriptive indices of air pollutants and climate factors in the city Tian** from January 1, 2017 to December 31, 2018 (reported as per day). Table 5: PLS-PM analysis for the relationships between latent variables.
Additional file 2
: Table 2. Total of DMR between patients with RSA and controls detected through RRBS.
Additional file 3
: Fig. 1. Cell communication analysis between 14 cluster cells. Circle plot shows the number of interactions and interaction weights/strength of 14 cluster cells between case and control.
Additional file 4
: Fig. 2. Molecular details and subclusters of dNKs were revealed by scRNA-seq. (A) A UMAP projection of the dNKs from one RSA patient and one matched healthy control. Different colors indicate cell clusters. (B) Dot plot shows the expression of marker genes for each subcluster of dNK. (C) Dot plot shows the expression of cell cycle-related genes. (D) Developmental trajectories of dNK subsets, cells colored by conditions of trajectories state, groups, subclusters, and pseudotime.
Additional file 5
: Fig. 3. Molecular details and subclusters of dM were revealed by scRNA-seq. (A) A UMAP projection of the dM from three RSA patients and three matched healthy controls. Different colors indicate cell clusters. (B) Dot plot shows the expression of marker genes for each subcluster of dM. (C) Dot plot shows the expression of cell cycle-related genes. (D) Developmental trajectories of dM subsets, cells colored by conditions of trajectories state, groups, subclusters, and pseudotime.
Additional file 6
: Fig. 4. Analysis of IGF2BP1 expression and its promoter region. (A) ROC analysis of methylation of IGF2BP1 in patient with RSA and controls. (B) scRNA-seq analysis of the expression of IGF2BP1 in 14 cell subsets. (C) Distribution of CpG island in the IGF2BP1 promoter region. (D) Prediction of transcription factors binding to each CpG island.
Additional file 7
: Fig. 5. The relationship between air pollution-related genes and RSA. (A) and (B) Monthly air pollutant concentration curve from 2014 to 2018. (C) Correlation heatmap plot shows the association between six air pollutants.
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Zhu, W., Gu, Y., Li, M. et al. Integrated single-cell RNA-seq and DNA methylation reveal the effects of air pollution in patients with recurrent spontaneous abortion. Clin Epigenet 14, 105 (2022). https://doi.org/10.1186/s13148-022-01327-2
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DOI: https://doi.org/10.1186/s13148-022-01327-2