ABSTRACT Cesarean section (CS) is recognized as being a shared environmental risk factor associated with chronic immune disease. A study of maternal gene expression changes between different

delivery modes can add to our understanding of how CS contributes to disease patterns later in life. We evaluated the association of delivery mode with postpartum gene expression using a

cross-sectional study of 324 mothers who delivered full-term (≥ 37 weeks) singletons. Of these, 181 mothers had a vaginal delivery and 143 had a CS delivery (60 with and 83 without labor).

Antimicrobial peptides (AMP) were upregulated in vaginal delivery compared to CS with or without labor. Peptidase inhibitor 3 (PI3), a gene in the antimicrobial peptide pathway and known to

or without labor. Further exploration is needed to determine if AMP upregulation provides protection against CS-associated diseases later in life. SIMILAR CONTENT BEING VIEWED BY OTHERS

PERSISTENCE OF BIRTH MODE-DEPENDENT EFFECTS ON GUT MICROBIOME COMPOSITION, IMMUNE SYSTEM STIMULATION AND ANTIMICROBIAL RESISTANCE DURING THE FIRST YEAR OF LIFE Article Open access 26 March

2021 IDENTIFICATION OF A NOVEL GENE SIGNATURE IN SECOND-TRIMESTER AMNIOTIC FLUID FOR THE PREDICTION OF PRETERM BIRTH Article Open access 31 March 2022 CERVICOVAGINAL NATURAL ANTIMICROBIAL

EXPRESSION IN PREGNANCY AND ASSOCIATION WITH SPONTANEOUS PRETERM BIRTH Article Open access 21 July 2020 INTRODUCTION While it is widely known that birthing a child is a physiologically

stressful event, the research surrounding how it affects the expression of genes in both mothers and infants is still sparse. Cesarean section (CS) is now recognized as being a shared

environmental risk factor associated with chronic immune diseases in the offspring1,2,3. At the cellular level, hematological and immunologic markers differ in the umbilical cord blood from

those born by vaginal delivery (VD) compared with CS, with higher numbers of leukocytes, including neutrophils, monocytes and natural killer cells detected in VD4,5. Children born by CS have

an increased risk for developing certain diseases later in life, such as asthma and allergies2,3,6,7,8,9, obesity10,11,12, connective tissue disorders1, inflammatory bowel disease1,2, type

I diabetes mellitus 13 and childhood leukemia14. Mothers who have given birth by CS have also been found to have a higher risk for developing an autoimmune disease as compared to those who

gave birth by VD15,16. Surgery may induce gene expression changes related to adverse outcomes. A recent study that examined changes in gene expression after major thoracoabdominal surgery

demonstrated that post-operatively, genes related to innate immunity and inflammation were upregulated while those related to adaptive immunity were downregulated17. One study thus far

identified epigenetic changes in the genome that occurred surrounding birth; more specifically infants born by CS had an increase in DNA-methylation in leukocytes compared to those born by

VD. It is not yet known if these epigenetic changes have a lasting effect on both mother and child18. Gene expression analysis of peripheral whole blood can provide an in-vivo perspective

into one’s response to physiological stressors and pathologic insults19. Furthermore, gene expression of patients with a variety of diseases correlates well with the status of the

disease20,21. As such, in an attempt to explore if gene expression changes vary by method of delivery, RNA sequencing data was examined in mothers at time of delivery. We hypothesized that

CS delivery (with or without labor) compared with VD is associated with gene expression changes in the mother, which could potentially contribute to disease patterns later in life in the

mother and offspring. RESULTS DEMOGRAPHIC AND CLINICAL DATA A total of 324 mothers with RNA sequencing available were included in the analysis. Of these, 181 had a VD and 143 had a CS

delivery (60 with and 83 without labor) (Table 1). Mothers undergoing CS without labor were older than those undergoing VD or CS with labor (mean age CS without labor 34.3 years vs. CS with

labor 31.4 years vs. VD 31.5 years; ANOVA p-value = 0.0002). Mothers undergoing CS without labor had a slightly lower gestational age at delivery than those having VD or CS with labor,

although all deliveries were full term (mean gestational age CS without labor 38.8 weeks vs. CS with labor 39.1 weeks vs. VD 39.3 weeks; ANOVA p-value = 0.001). For mothers who had a CS, the

indications for CS were also recorded (Table 1). For CS with labor, the top reason for CS was failure to progress or prolonged labor (N = 34) and for mothers undergoing CS without labor,

the top reason was repeat CS (N = 67). CS (WITH OR WITHOUT LABOR) VS. VAGINAL DELIVERY Samples from CS (with or without labor, N = 143) vs. VD (N = 181) were compared. Ninety-one genes were

found to be differentially expressed at FDR ≤ 0.05 and magnitude of log2(fold-change) ≥ 0.5 as highlighted in the volcano plot in Fig. 1A and listed in Supplementary Table S1. Of the

differentially expressed genes, 27 (30%) were upregulated in VD. Antimicrobial peptides were the top pathway and included PI3, which was the top gene upregulated in VD (Supplementary Table

S2). No genes were found to be differentially expressed between delivery modes in an independent analysis of prenatal samples from these mothers, thus confirming the changes did not exist

before delivery (lowest FDR > 0.99). CS WITH LABOR VS. VAGINAL DELIVERY Samples from CS with labor (N = 60) vs. VD (N = 181) were compared. Using a cutoff for false discovery rate (FDR) ≤

 0.05 and magnitude of log2(fold-change) ≥ 0.5, 506 genes were found to be differentially expressed (Fig. 1B, Supplementary Table S3). Of the 506 differentially expressed genes, 163 (32%)

were upregulated in VD and were enriched in the pathways listed in Table 2. Antimicrobial peptides and interferon signaling were among the top pathways and included the PI3 gene. Genes in

neutrophil degranulation and interleukin signaling pathways were upregulated in CS with labor. No genes were found to be differentially expressed in a separate analysis of only prenatal

samples (lowest FDR > 0.99). CS WITHOUT LABOR VS. VAGINAL DELIVERY Samples from CS without labor (N = 83) vs. VD (N = 181) were compared. Differential gene expression analysis resulted in

41 genes at FDR ≤ 0.05 and log2(fold-change) ≥ 0.5 (Fig. 1C, Supplementary Table S4). Of these, 26 (63%) were upregulated in VD and 294 (37%) were downregulated. Table 3 lists the pathways

obtained with at least 5 differentially expressed genes (separated by positive or negative fold change) in a pathway. Antimicrobial peptides and neutrophil degranulation were the top

pathways for genes upregulated in VD. PI3 was the top gene upregulated in VD. No significant pathways were found for genes upregulated in CS without labor. No genes were found to be

differentially expressed in an independent analysis of prenatal samples from these mothers (lowest FDR > 0.99). CS WITH LABOR VS. WITHOUT LABOR Samples from CS with labor (N = 60) vs. CS

without labor (N = 83) were compared. Differential gene expression analysis resulted in 263 genes at FDR ≤ 0.05 and log2(fold-change) ≥ 0.5 (Fig. 1D, Supplementary Table S5). Of these 263

genes, 60 (23%) were upregulated in CS without labor and 203 (77%) were upregulated in CS with labor. Table 4 lists immune pathways including interferon signaling and Cytokine signaling

genes which were downregulated, and neutrophil degranulation and interleukin signaling genes, which were upregulated in CS with labor. No genes were found to be differentially expressed in

the corresponding prenatal samples for the two groups (lowest FDR > 0.99). DIFFERENTIALLY EXPRESSED GENES AMONG ALL 3 GROUPS Likelihood Ratio Test (LRT) in the DESeq222 package was used

for identification of genes that are not constant across the three delivery modes. The top 50 genes with the highest fold-change in either direction were selected from DEGs obtained for the

two comparisons (VD vs. CS with labor, CS with labor vs. CS without labor) and all 41 DEGs were selected for CS without labor vs. VD. These genes were then intersected with 2,739 genes found

to show change in expression across delivery modes with LRT (adjusted p-value < 0.001). This led to 81 genes (Supplementary Table S6) that were used for clustering samples across the

three delivery modes (Fig. 2). PI3 was among the genes upregulated in VD compared to CS with or without labor (Fig. 3). While an independent analysis of prenatal samples did not show

elevated expression in mothers undergoing VD (Fig. 3A,B), PI3 expression was increased in VD compared to CS in post-delivery samples (Fig. 3C,D). DISCUSSION Differential gene expression was

found between the delivery modes of VD, CS with labor, and CS without labor. Of particular interest, there is an enrichment of antimicrobial peptides (AMP) pathways in VD. PI3 gene, a known

AMP, showed a twofold increase in VD (adjusted p-value < 10–9) compared to CS. Genes in the AMP pathways are upregulated in VD samples compared to both CS with or without labor. AMPs are

small molecular weight proteins that modulate host immune response against bacteria, viruses, and fungi23,24. Among the AMPs upregulated in VD compared to CS with or without labor (CTGS,

DEFA1, DEFA1B, DEFA3, DEFA4, ELANE, GNLY, GZMH, LCN2, LTF, PI3, PRTN3, RNASE3, SEMG1), PI3 was the most significant in terms of adjusted p-value (< 10–9) and fold change (> twofold).

The peptidase inhibitor 3 (PI3) gene encodes elafin and demonstrates antimicrobial and anti-inflammatory activities25. Elafin has been shown to play a protective role in inflammatory bowel

disease26, asthma27, and has been shown to be downregulated in acute stages of acute respiratory distress syndrome28. Elafin has also been proposed to be a modulator of innate immunity in

the lower genital tract29. Furthermore, elafin expression was found to be elevated in the cervix as a response to pathogens during preterm labor30. Given the apparent protective

antimicrobial and anti-inflammatory role of AMPs, we postulate that lower expression of PI3 in CS deliveries compared with vaginal deliveries, even if transient, may lead to may lead to an

inflammatory and immune cascade and play a role in the reported immune and inflammatory adverse outcomes associated with CS delivery for both mothers and

offspring1,2,3,6,7,8,9,10,11,12,13,14,15,16; this warrants further exploration. Other differentially expressed pathways between delivery modes involve the inflammatory and immune pathways

(neutrophil degranulation, interferon signaling). The significance of these pathways also require further investigation given the known stress and inflammation that occur both with labor31

and with major surgery17. Based on the known increased risk of chronic immune diseases in both mothers delivering by CS and their infants compared to VD, the finding of AMP upregulation,

particularly PI3, in VD compared to CS has potential clinical implications. If further research confirms this association and a causal relationship is found, this could lead to the

development of therapeutic interventions aimed to increase levels of AMPs in women undergoing CS. Our main strength is that we included a large number of subjects in this cohort of pregnant

women undergoing gene expression analyses from blood samples. Because maternal blood sample collection was performed prenatally and postpartum, we were able to independently analyze samples

from these two timepoints to conclude that changes in gene expression were influenced by the mode of delivery as no genes were differentially expressed across delivery modes in the prenatal

samples. A limitation of the study is that maternal prenatal blood was collected at various stages of the first and second trimesters between 12 and 27 weeks’ gestation (mean: 26.4 weeks,

95% confidence interval: 25.9–26.8). It is possible that confounding factors contributing to the reason a mother had a CS vs VD, including a pregnant woman’s medical and obstetric status and

fetal status may lead to changes in gene expression. Although many confounding factors associated with delivery mode were adjusted for in the analysis (maternal age, maternal BMI,

gestational age and computed ancestry), other factors were used as exclusion criteria for the analysis (multiple births, preterm births), and that no prenatal differences were seen in gene

expression between mothers who then went on to have CS vs. VD, it is possible other confounding factors may have played a role in differences seen. It is known that following the rupture of

membranes, there is an increased chance of ascending infections which may lead to increased AMP responses; in future studies, it may be valuable to account for if there was rupture of

membranes, and duration between rupture of membranes and delivery. In addition, the study is limited due to the lack of long-term follow up of subjects to assess the development of possible

adverse health outcomes. In conclusion, differentially expressed genes were found in mothers according to delivery mode with enrichment of antimicrobial peptide pathways in vaginal delivery

compared to both Cesarean section with or without labor. Further exploration is needed to confirm the differential expression seen, determine if similar changes are seen in gene expression,

or gene products, in the offspring, and identify longer term epigenetic changes including associations with long-term clinical outcomes in both mother and baby given the risk of diseases

associated with Cesarean section. MATERIALS AND METHODS SUBJECT ENROLLMENT This is a cross-sectional study carried out at Inova Fairfax Medical Center, Inova Health System, Falls Church,

Virginia. The study was designed to identify genomic, clinical, and environmental risk factors that may enhance our understanding of health outcomes. Data were collected on 324 mothers who

delivered from June 2013 to November 2014 and who had RNA sequencing available. Subjects were recruited during pregnancy at the time of their routine obstetric care visits. Only mothers with

full term (37 weeks–0 days or more) singletons were included. Emergency and urgent CS deliveries due to maternal and fetal indications were excluded because gene expression would be

expected to vary greatly across a variety of indications for time sensitive delivery. All participants provided written informed consent. The study was approved by the Western Institutional

Review Board (WIRB#20120204) and the Inova Institutional Review Board (Inova IRB#15-1804). All experiments were performed in accordance with relevant guidelines and regulations. DATA

COLLECTION Detailed demographic and clinical data were extracted from the medical record including: maternal age; pre-pregnancy body mass index (BMI); gestational age at delivery; delivery

mode (VD or CS); if CS was performed with labor (regular uterine contractions and cervical dilation) or without labor; and indication for CS. Data were collected from maternal questionnaires

completed prenatally including race and ethnicity. SAMPLE COLLECTION AND PROCESSING Peripheral blood was obtained from enrolled mothers within 2 days of delivery (between 1 and 39 h).

Prenatal blood samples (collected between 12 and 27 weeks’ gestation) were also available for these mothers. Blood was transferred into PaxGene (PreAnalytiX) tubes for total RNA isolation

with the QiaSymphony (Qiagen) using Ambion MagMax RNA Isolation kit (Thermo Fisher Scientific) per manufacturer's protocol. RNA was purified and concentrated using a ZR-96 RNA Clean

& Concentrator kit (Thermo Fisher Scientific) prior to quantification by NanoDrop spectrophotometry (Thermo Fisher Scientific). Total RNA was stored at – 80 °C until analysis. SAMPLE

SEQUENCING RNA samples were sequenced by the Illumina HiSeq2000 at Expression Analysis Inc. (Durham, North Carolina). For each sample, raw RNA reads in FASTQ format were aligned to hg19

using STAR 2.5.3a32. Standard quality control metrics were derived from the aligned reads using RNA-SeQC 1.1.933. Gene-level and transcript-level expression quantifications were performed

using RSEM 1.3.034 against the Gencode 26 reference transcript annotation35. Finally, expression results in expected counts and transcripts per million (TPM) of each sample were combined

into a single matrix using a customized Perl program. DIFFERENTIAL GENE EXPRESSION AND PATHWAY ANALYSIS EdgeR36 was used for differential gene expression and previously published guidelines

were followed for the analysis37. Genes with low counts were filtered out following EdgeR recommendation where at least n samples should have > 10/L counts per million where L is the

smallest library size and n is the number of samples in the smaller group. In summary, lowly expressed genes were filtered and library sizes were recalculated, followed by trimmed mean of

M-values (TMM) normalization (calcNormFactors), dispersion estimation using the negative binomial (NB) model (estimateDisp), extension of the NB model with quasi-likelihood (QL) methods

(glmQLFit), and finally, testing for differential gene expression (glmQLFTest). Whole genome sequencing (WGS) data were also available for these subjects; common single nucleotide variants

from WGS were used to compute principal components for depicting ancestry. Age, pre-pregnancy BMI, RNA sequencing processing batch, gestational age (in weeks), duration between birth and

sample collection, and the top 3 principal components derived from genomic sequencing were used as covariates in the analysis. In order to detect statistically significant changes across a

range of magnitude, a cutoff of absolute log2(fold-change) ≥ 0.5 and false discovery rate (FDR) ≤ 0.05 were used for selecting differentially expressed genes38. R package ggplot2 was used to

create volcano plots (Fig. 1), and density plots for PI3 expression across delivery modes (Fig. 3). Hierarchical clustering was performed and heatmap plots (Fig. 2) were generated using

heatmap.3 R package. Pathway analysis was performed using Reactome39, a manually curated pathway database that contains 12,608 human reactions organized into 2282 pathways involving 11,053

proteins as of version 71. Genes were separated by positive and negative fold-change in a given delivery mode for each pair-wise differential expression analysis and provided as input to

Reactome for pathway analysis. STATISTICAL ANALYSIS Comparisons of demographics and outcomes data were performed between women according to mode of delivery. Association between ethnicity

and delivery mode was tested with Fisher’s Exact Test, as implemented in the R40 function fisher.test(). One-way analysis of variance (ANOVA) was performed using R function aov() to test for

differences in age, BMI and gestational age across delivery modes. PI3 expression counts between delivery modes were compared using Student’s t-test, as implemented in R function t.test().

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Statistical Computing_ (R Foundation of Statistical Computing, Vienna, 2013). Google Scholar  Download references ACKNOWLEDGEMENTS Inova expresses its appreciation to the Fairfax County in

VA, which has supported Inova’s research projects with annual funding from its Contributory Fund (Fund 10030). Research reported in this publication was supported in part by the National

Institute of Child Health and Human Development under Award Number K23HD099240 (Hourigan). The content is solely the responsibility of the authors and does not necessarily represent the

official views of the National Institutes of Health. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Inova Health System, Falls Church, VA, USA Prachi Kothiyal, Xinyue Liu, Sahel Hazrati, 

Kathi Huddleston & Wendy S. W. Wong * Inova Children’s Hospital, Falls Church, VA, USA Keriann Schulkers & Suchitra K. Hourigan * Persona Biomed, Inc., Alexandria, VA, USA Thierry

Vilboux * Women’s Service Line and the Women’s Health Integrated Research Center, Inova Health System, Falls Church, VA, USA Luis M. Gomez, Thomas P. Conrads & G. Larry Maxwell * College

of Health and Human Services, George Mason University, Fairfax, VA, USA Kathi Huddleston * Departments of Surgery and Oncology, Johns Hopkins University School of Medicine, Baltimore, MD,

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J.N. and S.K.H. made substantial contributions to the conception or design of the work. P.K., K.S., X.L., S.H., T.V., L.G., W.S.W., T.C., L.M. and S.K.H. made substantial contributions to

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P., Schulkers, K., Liu, X. _et al._ Differences in maternal gene expression in Cesarean section delivery compared with vaginal delivery. _Sci Rep_ 10, 17797 (2020).

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