# Differential gene expression in bovine endometrial epithelial cells after challenge with LPS; specific implications for genes involved in embryo maternal interactions

Yongzhi Guo, Tom van Schaik, Naveed Jhamat, Adnan Niazi, Metasu Chanrot, Gilles Charpigny, Jean-François Valarcher, Erik Bongcam-Rudloff, Göran Andersson, Patrice Humblot

<figure><img src="image_1.png" /></figure>

## To cite this version:

Yongzhi Guo, Tom van Schaik, Naveed Jhamat, Adnan Niazi, Metasu Chanrot, et al. Differential gene expression in bovine endometrial epithelial cells after challenge with LPS; specific implications for genes involved in embryo maternal interactions. PLoS ONE, 2019, 14 (9), pp.e0222081. 10.1371/journal.pone.0222081. hal-02625155

HAL Id: hal-02625155

https://hal.inrae.fr/hal-02625155

Submitted on 26 May 2020

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH ARTICLE

# Differential gene expression in bovine endometrial epithelial cells after challenge with LPS; specific implications for genes involved in embryo maternal interactions

Yongzhi Guo<sup>1*</sup>, Tom van Schaik<sup>2,3</sup>, Naveed Jhamat<sup>2,4</sup>, Adnan Niazi<sup>5,6</sup>, Metasu Chanrot<sup>1,7</sup>, Gilles Charpigny<sup>8</sup>, Jean Francois Valarcher<sup>9</sup>, Erik Bongcam-Rudloff<sup>5</sup>, Göran Andersson<sup>2</sup>, Patrice Humblot<sup>1</sup>

<figure><img src="image_3.png" /></figure>

1 Swedish University of Agricultural Sciences, Department of Clinical Sciences, Division of Reproduction, Uppsala, Sweden, 2 Swedish University of Agricultural Sciences, Department of Animal Breeding and Genetics, Section of Molecular Genetics, Uppsala, Sweden, 3 Department of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands, 4 Department of Information Technology, University of the Punjab, Lahore, Pakistan, 5 Swedish University of Agricultural Sciences, Department of Animal Breeding and Genetics, Section of Bioinformatics and SLU Bioinformatics Infrastructure, Uppsala, Sweden, 6 Science for Life Laboratory, Uppsala University, Uppsala, Sweden, 7 Faculty of Veterinary Science, Rajamangala University of Technology Srivijaya, Nakorn Sri Thammarat, Thailand, 8 INRA, UMR1198 Biologie du Développement et Reproduction, Jouy-en-Josas, France, 9 Swedish University of Agricultural Sciences, Department of Clinical Sciences, Division of Ruminant Medicine, Uppsala, Sweden

* yongzhi.guo@slu.se

<figure><img src="image_4.png" /></figure>

OPEN ACCESS

**Citation:** Guo Y, van Schaik T, Jhamat N, Niazi A, Chanrot M, Charpigny G, et al. (2019) Differential gene expression in bovine endometrial epithelial cells after challenge with LPS; specific implications for genes involved in embryo maternal interactions. PLoS ONE 14(9): e0222081. https://doi.org/10.1371/journal.pone.0222081

**Editor:** Peter J. Hansen, University of Florida, UNITED STATES

**Received:** May 29, 2019

**Accepted:** August 21, 2019

**Published:** September 5, 2019

**Copyright:** © 2019 Guo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** Our RNAseq raw data were uploaded to Nucleotide Sequence Database (ENA) with the accession number PRJEB34011.

**Funding:** This work was performed with the financial support from the FP7 EU project "PROLIFIC" (Grant KBBE 311776) to PH and FORMAS (Grant 2015-888) to PH and by a grant from Rajamangala University of Technology Srivijaya (RMUSTSV), Thailand to MC.

## Abstract

Lipopolysaccharide (LPS) expressed on the surface of Gram-negative bacteria activates pro-inflammatory pathways, dys-regulates the function of endometrial cells and is a key player in the mechanisms involved in endometritis. This study aimed to investigate the effects of LPS on bovine endometrial epithelial cells (bEEC) from whole transcriptome with a special focus on genes involved in embryo-maternal interactions. Following *in vitro* culture, bEEC from three cows were exposed to 0, 2, and 8 µg/mL LPS for 24h. RNA samples extracted at 0 and 24 hours were analyzed by RNA sequencing (RNA-seq). At 24h, 2035 differentially expressed genes (DEGs) were identified between controls and samples treated with 2 µg/mL LPS. Gene ontology analysis showed that over-expressed DEGs were associated to immune response, response to stress and external stimuli, catalytic activity, and cell cycle. Genes associated with cell membrane and cell adhesion pathways were under-expressed. LPS induced changes in expression of specific genes related to embryo-maternal interactions including under-expression of eight members of the cadherin superfamily, over-expression of six members of the mucin family, and differential expression of a large set of genes binding the above molecules and of more than 20 transcripts coding for cytokines and their receptors. Type I interferon-τ dependent genes were also over-expressed. From a sub-set of 19 genes, (biological replicates of bEEC from cows taken at time 6 (n = 3), 24 (n = 6) and 48 hours (n = 3), and 2 technical replicates per sample) differential gene expression was confirmed by RT²-qPCR (r² between fold changes at 24 hours by RT²-qPCR and RNA-seq = 0.97). These results indicate that LPS affects the function of bEEC in many ways by differential transcription, glycolytic metabolism and oxidative stress. Many
**Competing interests:** The authors have declared that no competing interests exist.

transcriptomic signatures related to implantation and embryo maternal interactions were strongly affected by LPS. These results pave the way for further studies to investigate the duration of these changes and their possible impact on endometrial function and fertility.

Introduction

Escherichia coli (E. coli) is commonly associated with uterine infection in post-partum cows [1, 2]. Lipopolysaccharide (LPS), at the outer membrane of E. coli and other Gram-negative bacteria, is one of the most powerful bacterial virulence factors triggering inflammation of the endometrium [3, 4]. Moreover, LPS has been reported to induce implantation failure, and pregnancy losses in the human species [5, 6]. LPS binding to toll-like receptors trigger pathways activating the production of pro-inflammatory cytokines [7, 8]. In addition, in different cell models including epithelial cells from different species, oxidative stress and glycolysis associated also to increased proliferation have been reported [9]. Cytokines including growth factors and their corresponding receptors expressed on endometrial cells play an important role in the processes regulating embryonic development, and in the interactions between the embryo and maternal cells, especially at time of implantation [10]. Successful implantation is the result of a very precise balance between cytokines which are necessary to the induction of uterine remodeling (such as LIF, IL-6, IL-17) and cytokines such as TGFβ, IL-10, IL-27, G-CSF limiting the inflammatory processes and driving the response of the maternal endometrium to embryo towards immune-tolerance [11]. In human and mice, it has been shown that other molecules such as Galectin-1 (Gal-1) are involved in the regulation of the differentiation of naïve CD4+ T cells to CD4+CD25+ regulatory T (Treg) cells, which are key effectors in orientating the innate immune response mechanisms towards immune-tolerance [12-14].

In ruminants, due to essential for maternal recognition of pregnancy and maintenance, a special attention has been paid to IFN-τ [15, 16]. Transcriptomic and functional studies in both the ewe and cow have shown that a large set of genes are over-expressed in the endometrium in response to IFN-τ [17]. Some of these genes such as MX1 and MX2 encode proteins that are also involved in the response to pathogens or disease [18].

Deviations in the elevation of pro-inflammatory cytokines in the uterine environment during the preimplantation period may damage endometrial cells, perturb embryo maternal interactions, and trigger DNA damage in embryonic cells. All these processes are potentially involved in implantation failures [5, 19].

More recently, with similar tools, the *in vitro* effects of LPS treatment on gene expression patterns were studied in primary cultures of mixed bovine epithelial and stromal endometrial cells, and gene expression of *SAMD9*, *PLAC8* and *LGALS9* which encode proteins that are important for early pregnancy were over-expressed [20, 21].

In this study, we used RNA-sequencing technology to analyze with an unbiased approach, gene expression changes associated to the response of a pure population of bovine endometrial epithelial cells to a single challenge with *E coli* LPS. The data obtained here provide additional results on gene expression changes as a complement to former published information on the phenotypic response in terms of proliferation and survival of cells [9] and on the changes in proteomic profiles induced by LPS [22] in the same cell model. The present results show a wide range of responses of these cells to LPS, confirming the high pro-inflammatory role of this molecule and former information obtained on the corresponding pathways in endometrial cells and other types of epithelial cells [20, 23]. In addition, we provide novel information
regarding the impact of LPS on sets of specific genes related to embyromaternal interactions and corresponding pathways which were here specifically scrutinized. Although incomplete due to the nature of this model based on a single cell type, these results contribute to understand the potential role of LPS on persistence of inflammation in the endometrium and its possible unfavorable effects on endometrial function possibly impairing fertility.

## Materials and methods

### Isolation of bovine endometrial epithelial cells (EEC)

The uterine horns from three Swedish Red Breed (SRB) cows were collected from slaughterhouse (Lövsta SLU, Uppsala, Sweden). Endometrial epithelial cells were separated then cultured according to procedures previously described [9]. Within one hour after slaughter, the endometrium was dissected into 5–6 cm long and 4–5 mm depth pieces. Tissue pieces were incubated for two hours at 39°C with collagenase IV (C5138, Sigma, Saint Louis, MO, USA) and hyaluronidase (250 U/mL) (H3506, Sigma) in PBS containing 2% BSA (Sigma). The suspension was then filtered through 250 µm gauze to remove mucus and undigested tissue. The filtrate was then passed through a 40 µm nylon sieve, which allowed the fibroblasts and blood cells to pass through while epithelial cells were retained. Epithelial cells were collected from the filter by backwashing with 30 mL PBS. Cells were centrifuged at 170 ×g for six minutes and the pellet re-suspended in three mL of PBS. Pellets were dispersed into a single cell suspension by passing through a fine gauge needle (20G). Cells were then cultured in F-12 medium (Dulbeco's modified eagle's medium, D6434, Sigma) containing 10% Fetal Bovine Serum (FBS), 1% Penicillin/streptomycin (5000 units/mL penicillin/streptomycin, Gibco, Carlsbad, CA, USA), 2 mM L-glutamine, 0.5% Liquid Media Supplement (ITS), and gentamycin (5 µg/mL) and nystatin (100 U/mL). Cells were seeded into a 25 cm² ventilation flask. Cell cultures were kept in a water-jacked incubator with 5% CO₂ at 39°C. Medium was changed every 1–2 days. Sub-cultivations were performed when epithelial cells attained 80 to 90% confluence [24, 25].

The purity of bovine endometrial epithelial cell (bEEC) culture was estimated by morphological observation following three to five passages. Potential residual contamination of fibroblasts was eliminated between each passage by detaching with 3 mL trypLETM express (Gibco 12605) for two min at 39°C, and aspiration of the supernatant. Cells retained in the bottom were then viewed under the microscope to determine the presence of fibroblasts [26]. After passage 3 and before LPS challenge, no fibroblasts were found by microscopic observation (Fig 1A). The purity of the epithelial cell culture was checked by flow-cytometry labelling cytokeratin (Primary Anti-cytokeratin 18 Ab, Abcam, Cambridge, UK, cat ab 668, and secondary Anti-alex 488 Ab, cat ab175473 used following manufacturer's instructions). From passage 2 and thereafter, more than 98% of cells expressed cytokeratin, confirming the very high purity of the cell culture system (Fig 1B).

### LPS challenge and Isolation of total RNA

As previously described [22], bEEC were challenged on passage 5, with 0, 2, and 8 µg/mL LPS from E.coli (O111:B4; Sigma). These concentrations of LPS reflected concentrations found in uterine fluid in cases of clinical endometritis [27, 28]. Moreover, we did choose to perform this study with these LPS dosages and a pure population of endometrial epithelial cells as used in our former experiments, in cell survival, proliferation, cytokine production and proteomic profiles 72 hours post LPS exposure [22]. At the time of challenge and 24 hours later, cells were detached by 5 mL trypLETM express (Gibco 12605) and washed with DPBS (Gibco). The present study in changes in gene expression by RNAseq and in subsequent validation studies were performed at earlier stage when compared to cell survival and proteomics, which
<figure><img src="image_5.png" /><figcaption>**Fig 1. Morphology and purity of bovine endometrial epithelial cells.** A. Cell morphology of bovine endometrial epithelial cells (bEECs) B. Representative Flow cytometric histogram of bEECs expressing the epithelial-specific cytokeratin-18 showing their appearance to the epithelial cell population. (98% of cells are in M1 region corresponding to this population).</figcaption></figure>

https://doi.org/10.1371/journal.pone.0222081.g001

corresponded also well with the design of previous studies showing changes in expression of TLR4 mRNA in bovine endometrial cells [29, 30]. Samples of two million cells were frozen at -80°C. Total RNA was extracted with the Allprep DNA/RNA/miRNA Universal Kit (Qiagen, Darmstadt, Germany, Cat. No. 80224), and the respective concentrations of total RNA and mRNA (RIN) was determined from Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). RNA samples had 28S/18S ratios ranging from 1.8 to 2.0, concentrations from 800–1580 ng/µl, and RIN values of RNA ranged from 8.8 to 10.0 (S1 Table).

## Library preparation and sequencing

Sequencing libraries from 12 samples (for each cow, on Time 0, and with 0, 2, and 8 µg/mL LPS at 24h, Fig 2) were prepared by the sequencing platform (Science for Life Laboratory, Uppsala University; https://www.scilifelab.se/) from 1µg of total RNA using the TruSeq stranded mRNA sample prep kit including polyA selection (RS-122-2101/2, Illumina Inc, San Diego, CA, USA) (15031047, revE). RNA sequencing was then performed using the Illumina HiSeq2500 system. Paired-end 125bp reads were obtained with sequence depth of about 30 million reads per sample.

## Data quality control and read abundance calculation

The quality of the RNA-Seq data generated from control (control time 0, n = 3 and control 24h, n = 3) and LPS-treated samples (2 µg/mL, n = 3 and 8 µg/mL, n = 3) were first studied using FastQC 0.11.2 [31]. The adapter sequences and low quality reads were then filtered with Trimmomatic 0.32 [32], and reads were mapped to the *Bos Taurus* reference genome *bosTau6* with STAR 2.4.0 software [33]. Of the filtered reads, approximately 97% were mapped to the *Bos taurus* reference genome. Uniquely mapped reads were counted to ENSEMBLv78 annotations with HTSeq 0.6.1 for downstream differential expression analyses [34]. The RNA-Seq experiment generated an average of 26.35 ± 1.96 (mean ± SD) million uniquely mapped reads per sample (S1 Table).

## Principal component analysis (PCA) and identification of significantly differentially expressed genes (DEGs) and pathway analysis

PCA and the identification of DEGs were performed with DESeq2 version 1.6.3 [35]. For the PCA, raw counts were normalized and log transformed with log function provided in
<figure><img src="image_6.png" /><figcaption>**Fig 2.** Comparison of differentially expressed genes. A. Design of comparisons made between groups (shown above) and Venn diagram of differentially expressed genes (DEGs) between each LPS-treated groups and controls (below). B. Venn diagram including the distribution of DEGs found in both LPS treatments together (combined 2 and 8 µg/mL LPS) versus control cells showing that most of the genes which were not common in A expressed similar changes as becoming DEGs in the “combined” comparison. C. Correlation of log2 fold changes between the 2 and 8 µg/mL LPS-treatment. D. MA-plot of all DEGs identified in the different LPS treatments, log2 fold changes for each gene between two samples (on the y-axis) versus the mean expression signal (on the x-axis). Blue (2µg/ml LPS), green (8 µg/ml LPS) and red dots (common) represent the genes with significant differential expression (Adjusted P value <0.05 from Benjamini Hochberg correction). Significant mean positive log2 fold changes corresponds to increased expression, whereas negative values corresponds to decreased expression. E. Principal Component Analysis (PCA) from the top 500 most variably expressed genes at 24h post LPS. Symbols for each cow have a different shape and with a different color for each condition as indicated to the right. Axis 1 reflects individual variation in basic gene expression between individuals whereas axis 2 reflects the effect of LPS treatment.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0222081.g002

DESeq2. After differential expression analysis, the resulting p-values were adjusted for multiple testing using the Benjamini-Hochberg procedure. DEGs with adjusted *p*-values < 0.05 were regarded as significant. DEGs were identified from analysis of paired wise comparisons (each cow individual untreated samples from which cell culture was issued taken as its own control) *i*) between control at time 0 and control at 24h, *ii*) between control at 24h vs 2 µg/mL and 8 µg/mL LPS-treated at 24h (LPS dosages taken individually), *iii*) between 2 µg/mL and 8 µg/mL LPS-treated samples at 24h, *iv*) between control samples and LPS-treated samples at 24h (2 and 8 µg/mL LPS taken together), (Fig 2A). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were determined using DAVID 6.7 [36, 37] with all Ensembl genes as background [38]. For GO term analysis, the DEGs were analyzed by running queries for each DEG against GO database, which provides information related to three ontologies: biological process (BP) cellular component (CC), molecular function (MF).
MA-plots are used to visualize the differential gene analysis results (a scatter plot of log₂ fold changes for each gene between two samples (on the y-axis) versus the mean expression signal (on the x-axis)). Consequently, genes with similar expression levels in two samples appear around the horizontal line y = 0. Analysis of genes possibly involved in disease and physiological system functions was based on GeneCards database (http://www.genecards.org/) which integrates gene-centric data from around 125 web sources.

## RT²-qPCR validation and changes in gene expression with time

Validations of relative gene expression levels obtained from RNA-seq were performed by RT²-qPCR (RT² Profiler™ PCR Array: CAPB13612, QiaGen, Hilden, Germany), using a subset of samples from those used for the RNA sequencing (Control 24 hours and 24 hours after 2 µg/mL LPS) and another set of samples issued from the culture of cells from 3 additional cows. Cells from these cows were exposed to 2 µg/mL LPS for 6, 24 and 48 hours and comparisons made with controls obtained at the same time. Two technical replicates were done on each biological replicate of bEEC from cows taken at time 6 (n = 3), 24 (n = 6) and 48 hours (n = 3) following LPS exposure. Nineteen different genes (*IFIT3*, *MUC1*, *IFIT1 MUC13*, *ITGB3*, *ITGB6*, *CDH26*, *PKP1*, *STAT1*, *NFKB1*, *SMAD3*, *LIF*, *MMP1*, *MMP7*, *MMP13*, *TGFB2*, *LGALS1*, *LGALS3*, and *LGALS9*) were selected for validation based on their biological relevance and on their significance to cover the full range of differential expression observed from RNAseq. When several genes from the same family were differentially expressed we took one member from list from highly significant ones and one member for which the adjusted value was close to 0.05. Results were normalized from changes for three housekeeping genes used as reference (*TBP*, *ACTB* and *GAPDH*). As differences were expressed the same way with the 3 reference genes, results are presented while using *ACTB* as reference.

## Statistical analyses

Generalized linear model was fitted and Wald test was performed to determine which of the observed fold changes were significantly different between pairwise analyses of controls and treatment groups. The ANOVA-like DESeq2 likelihood ratio statistic (LRT) test was used to test multiple levels of control and treatment at once. Adjusted p values *p* < 0.05 were considered to determine DEGs.

General linear models were used (proc GLM, SAS Ver 9.4) to analyze RT²qPCR data. Cow and time (6, 24, 48 hours) were included as main effects in the models potentially influencing the differences in expression between control vs 2 µg/mL LPS (y = expression LPS time x- expression Control time x) and ratio of expression; z = (expression LPS time x-expression Control time x) / expression Control time x). In this case, the paired wise statistics compares the observed value "y" or "z" to "0" to be observed in case of the "null hypothesis" (lack of effect). As the effects were systematically the same for the 2 above variables only "z" results are presented. When necessary log transformation was used to normalize the expression data for some genes. For the sake of clarity, non-transformed ratios are presented. The significance of individual comparisons between times were subsequently analyzed by using multiple comparison adjustments ("Scheffé" test and contrast option under GLM).

The correlation between fold changes obtained from RNA-seq and those from the RT²-qPCR experiment for the 19 genes, (mean fold change from 3 cows at 24 hours following 2 µg/mL LPS from the two techniques) was studied by using Spearman correlation coefficient.
# Results

## Overall differential gene expression analysis

The number of differentially expressed genes (DEGs) between control and LPS groups at 24 hours post-challenge are presented in Fig 2A. Overall, 2035 and 2073 DEGs were identified between control cells and cells treated with 2 and 8 µg/mL LPS, respectively. No gene was identified as being differentially expressed between the two different LPS dosages employed, allowing the two LPS-treated samples from each cow to be considered as biological replicates in some of the analyses. A total of 1748 DEGs were found to be common between the two treatment groups. Nearly 700 additional DEGs were identified when comparing both LPS treatments taken together (groups of 2 and 8 µg/mL LPS) to controls indicating that in almost all cases, gene expression levels for these genes show similar variation in response to LPS for both treatment groups (Fig 2B). This is further confirmed by the correlation coefficient between fold changes observed with 2 and 8 µg/mL LPS is r² = 0.99 (Fig 2C).

A slightly higher number of genes were identified as being over-expressed than under-expressed. From the 2035 genes significantly de-regulated 24h after exposure to 2µg/ml E.coli LPS, 1066 were over-expressed (52.4%), and 969 under-expressed (47.6%). Furthermore, higher fold changes were in general observed for the over-expressed genes when compared to genes with reduced expression levels (Fig 2D). DEGs, which were found as significant only for one of the LPS dosages (for the 2 and 8 µg/mL LPS, respectively) were close to the significance cut off level (*p* < 0.05). Due to the large common number of DEGs found and to the high degree of similarity between transcriptomic profiles observed between the two dosages (r² = 0.99; Fig 2D), the detailed analysis and interpretation was based mainly on the comparison between control vs 2 µg/mL LPS at 24h. The common response of LPS groups when compared to controls was further revealed from the principal component analysis (PCA) made from the top 500 genes displaying highest variability across samples (Fig 2E). Despite that bEECs obtained from one cow displayed an atypical basic expression pattern in controls, the PCA shows that response to LPS treatment appeared very consistent with the differential gene expression observed for the two other cows.

## Gene ontology analysis of the DEGs

DAVID 6.7 was used to identify biological pathways, through associated over-represented biological GO-terms corresponding to significantly DEGs in cells exposed to LPS. GO enrichments of the DEGs were categorized into 484 functional groups with a corresponding adjusted *p* value < 0.001. This total included 274 over-expressed and 210 under-expressed groups. For the over-expressed groups, 214 (78.1%), 15 (5.5%) and 45 (16.4%) were categorized into biological process (BP) cellular component (CC), molecular function (MF), respectively. For the under-expressed groups, 134 (64%), 52 (25%), and 24 (11%), were categorized into BP, CC, and MF, respectively (S2 Table). The dysregulated pathways with the most significant adjusted *p* values are illustrated in Fig 3. Gene ontology analysis showed that four out of the top 10 significantly overrepresented pathways were related to innate immune response, inflammatory response and antigen processing and presentation (Fig 3A, S1 Fig and S3 Table). Response to stress and external stimuli, transcription, catalytic activity and glycolysis were also highly over-represented. The top five underrepresented GO-terms are related to cell structures such cytoskeleton, cell membrane, binding and organelle (Fig 3B and S4 Table).

## KEGG pathway analysis of the DEGs

Overall, the DEGs were significantly enriched in 22 different KEGG pathways (adjusted *P*-values < 0.05) including 11 of overrepresented pathways and 11 of underrepresented pathways
A. Overrepresented (GO-terms)

<figure><img src="image_7.png" /></figure>

C. Overrepresented (KEGG-pathways)

<figure><img src="image_8.png" /></figure>

B. Underrepresented (GO-terms)

<figure><img src="image_9.png" /></figure>

D. Underrepresented (KEGG-pathways)

<figure><img src="image_10.png" /></figure>

**Fig 3.** Gene ontology (GO) and KEGG pathways analysis of over-expressed and under-expressed DEGs. A. Overrepresented GO-terms pathways, + = pathways related to immune function. B. Underrepresented GO-terms pathways C. Overrepresented KEGG pathways D. Underrepresented KEGG pathways. Vertical red dotted bars indicate the cut-off level for significance (P < 0.05). P-values were adjusted by Benjamini-Hochberg correction for multiple testing.

https://doi.org/10.1371/journal.pone.0222081.g003
(S5 and S6 Tables). Similarly to the above, six out of 10 overrepresented pathways were related to inflammation (Nucleotide-binding, oligomerization domain (NOD)-like receptor signaling pathway with 16 DEGs enriched, Toll-like receptor signaling pathway (19 DEGs), cytokine-cytokine receptor interaction (25 DEGs), antigen processing and presentation (12 DEGs), chemokine signaling pathway (23 DEGs), and apoptosis (19 DEGs) (Fig 3C). Three out of 10 of the underrepresented pathways were related to focal adhesion, regulation of actin cytoskeleton and adherent junction, which are protein complexes that are present at cell-cell junctions in epithelial and endothelial tissues (Fig 3D).

Comparing results from the present RNA-seq data to GeneCards database (http://www.genecards.org), we found different numbers of DEGs participating in acute inflammation (410 DEGs), innate immunity (441 DEGs), immune tolerance in pregnancy (120 DEGs), allergy (153 DEGs), and cell adhesion (626 DEGs). As embryo implantation is associated to inflammatory processes we found here also a quite high number of genes corresponding to this process through this classification (118 DEGs) (Fig 4 and S7 Table). Twenty four common DEGs (EDN1, TGFB2, TGFB3, FAS, C3, ICAM1, CXCL8, IL1RN, TP53, TNF, IL1A, CAT, F5, NFKB1, IL1B, PTGS1, CDKN2A, IL1R1, CCL2, ADA, IL6, CSF2, MMP1, and MMP9) were involved in all the above indicated biological processes. For these functions more over-expressed genes were found when compared to under-expressed ones as shown by the unbalanced ratios (S7 Table). A large number of DEGs were also associated to several physiological system functions and molecular and cellular functions. For cell skeleton (466 DEGs), cell proliferation (880 DEGs), cell apoptosis (760 DEGs) and signal transduction (755 DEGs) a large number of genes are identical to GeneCards database. On the contrary to the above, for these functions the number of over- and under-expressed genes are similar and the corresponding ratios are close to one (S7 Table).

Based on the results described in S7 Table, the list of the top 10 most significantly over- and under-expressed genes with respect to immune function, inflammation, infection and disease, are listed in S8 Table. Some of these genes and their products have a pivotal role for cells in response to disease as shown by their presence in several of the corresponding above processes (S8 Table). In particular, CX3CL8 was involved in all of 10 biological processes. Very high proportions are also observed for CX3CL1 (8/10), TNF (8/10), CXCL6 (5/10), C3 (9/10), IL1A (7/10) and LGALS9 (6/10) over-expressed. Similarly high proportions are observed for some of the under-expressed genes such as PTHLH (6/10) and TIMP3 (5/10).

S9 Table provides the top 100 most significantly over- and under-expressed genes by log2 fold-change in response to 2 µg/mL of LPS treatment. Among the 19 most significantly over-expressed genes (> 3 fold), 7 were associated with inflammation (CXCL6, BCL2A1, LGALS9, C3, BIRC3, CFB, and CD40) and 9 out of 19 genes were associated with infection (CXCL6, BCL2A1, LGALS9, C3, BIRC3, CFB, CD40, CTSC and TCN1). On the contrary, among the six most under-expressed genes (>1.5-fold), no DEGs were known to be related to inflammation.

Genes of the bovine leukocyte antigen (BoLA) which are part of the Major Histocompatibility Complex (MHC) of cattle were over-expressed. We found that both genes encoding class I (BOLA-A, BOLA-NC), and class II molecules (HLA-DMB, BOLA-DQA1, and BOLA-DRA) were over-expressed after LPS-treatment (Fig 5A, and S1B Fig).

## Genes related to implantation and maternal response to the embryo

A large number of genes encoding for proteins belonging to several families of molecules involved in embryo maternal interactions and implantation were differentially expressed in response to LPS treatment. This includes genes encoding molecules involved in cell structure (actins, actinin), calcium metabolism and membrane properties (calcitonin), cell adhesion
<figure><img src="image_11.png" /><figcaption>**Fig 4. Comparison of DEGs after challenge with LPS to GeneCards database.** Numbers of DEGs participating in acute inflammation (410 DEGs), innate immune (441 DEGs), immune tolerance in pregnancy (120 DEGs), allergy (153 DEGs), cell adhesion (626 DEGs) and embryo implantation (118 DEGs). The figure illustrates the relatively high number of DEGs common to two pathways and 24 common DEGs were involved in all above mentioned biological processes (http://www.genecards.org/).</figcaption></figure>

https://doi.org/10.1371/journal.pone.0222081.g004

(catenins, plakophilin, cadherins, integrins), protection of epithelium (mucins) and enzymes such as Matrix Metallo Peptidase (MMPs) controlling tissue-remodeling.

Four members of the cadherin superfamily which are transmembrane glycoproteins that mediates cell-cell interactions through calcium binding (i.e. *ITGB6*, *CDH26*, *ITGAV* and *CELSR1*) were over-expressed after LPS-treatment. In addition, four integrin-family transcripts (*ITGB3*, *ITGB4*, *ITGB5*, and *ITGA7*) and most of transcripts coding for cell adhesion molecules (for instance, *CTNNA3*, *CTNNAL1*, *CDH2*, *PCDH7*, *CT*, *PKP1*, *PKP4*) showed lower mRNA levels after treatment by LPS (Fig 5B). Furthermore, we found that many genes of the mucin family (*MUC1*, *MUC4*, *MUC13*, *MUC16*, *MUC20*, and *F1MUC1*) and genes of matrix metalloproteinase (MMPs) family (*MMP1*, *MMP7*, *MMP9*, *MMP13*) were strongly over-expressed. Consistent with the above results, their inhibitor *TIMP3* was shown to be strongly under-expressed after LPS-treatment (Fig 5C).

A large number of interferon-τ (IFNT)-induced genes were all over-expressed after LPS-treatment (Fig 5D). These include *IFIT1*, *IFIT2*, *IFIT3*, *IFIT5*, *IFITM2*, *IFITM3*, *PARP12*, *ZNFX1*, *HERC6*, *RNF213*, *CXCR7*, *DDX58*, *PLAC8*, *RSAD2* and *STAT1*. Genes of galectin family were also significantly de-regulated. For instance, Gal-9 (*LGALS-9*), which has been related
<figure><img src="image_12.png" /><figcaption>**Fig 5.** Mean Log2 fold changes (mean ± sem) for DEGs related to implantation and maternal response to the embryo. A. Changes in mRNA expression for genes of the bovine leukocyte antigen molecules (BoLA) family. B. Changes in expression for genes related to cell adhesion. C. Changes in expression for mucins, integrins and metalloproteinase genes. D. Changes in expression for interferon Tau induced genes. E. Changes in expression for genes of the galectin family. All changes significant *p* < 0.05 from adjusted *p* value.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0222081.g005

to response infection is strongly over-expressed. On the contrary, *LGALS-1* and *LGALS-3* were under-expressed in both LPS groups vs controls (Fig 5E).

## Genes related to methylation and acetylation

In addition, significant changes in expression for a set genes susceptible to influence the methylation and acetylation profiles of cells were found. Most particularly, MBD4 which binds de novo methyl transferases (DNMTs) to DNA and a number of genes from the methyl transferase family i.e. METTL9 (methyltransferase like 9), METTL22 (methyltransferase like 22), TRMT1 (TRM1 tRNA methyltransferase 1 homolog) were under-expressed whereas two members of the lysine demethylase family (KDM2 and KDM7) were overexpressed. Concomitantly, three members of the Histone Deacetylase family (i.e. HDAC4, 5 and 6) were under-expressed, whereas the acetyl transferases (ACAT2) was over-expressed. Inhibition of HDAC induces transcriptional repression of high copy number genes in breast cancer [39] and ACAT2 gene encode enzymes involved in lipid metabolism [40].
RT²-PCR validation and temporal changes in gene expression induced by LPS

RT²-qPCR validation study of 19 differentially expressed genes (over or under expressed) from RNA-seq showed that all tested genes varied significantly and in the same way with the two methods. Notably, all DEGs with an adjusted p value close to 0.05 from RNAseq were confirmed as differentially expressed by the RT²-qPCR validation. Moreover, the correlation coefficient between fold changes obtained by RNA-seq and RT²-qPCR from samples obtained at 24 hours for the 2 µg/ml LPS dosage was very high with a r² value close to 1 (Table 1). Gene-expression profiles at 6, 24, and 48 hours revealed differences in patterns between groups of genes. Genes related to acute inflammation and signal transduction pathways such as *IFIT1*, *IFIT3*, *ITGB6*, *NFKB*, *MMP13* and including transcription factors such as *LIF* and *STAT1* expressed a maximum increase at 6 hours and gradually dropped at 24 and 48 hours. For *MUC13*, *CDH26* and *MMP7* gene expression continuously increased from 6 to 48 hours whereas *LGALS9*, *SMD3*, *MUC1* and *MMP1* shown a higher expression at 24 hours followed by a decrease. For under-expressed genes, the expression profiles shown that *LGALS1*, *ITGB3* and to a lesser extent *PKP1* were decreasing continuously from 6 to 48 hours, whereas *LGALS3*, and *TGFB* presented low expression at 6 and 24 hours, and were closer to control levels at 48 hours (Fig 6).

<table><caption>Table 1. Fold changes obtained by RNA-seq and RT2-qPCR from samples obtained at 24 hours for the 2 µg/ml LPS dosage.</caption><thead><tr><th rowspan="2">Gene ID</th><th colspan="2">Fold changes (2 µg/ml vs control, 24h)</th></tr><tr><th>RNA-seq</th><th>RT²-qPCR RQ</th></tr></thead><tbody><tr><td><i>STAT1</i></td><td>1.51</td><td>1.93</td></tr><tr><td><i>LGALS9</i></td><td>11.25</td><td>28.17</td></tr><tr><td><i>NFKB1</i></td><td>2.41</td><td>2.36</td></tr><tr><td><i>SMAD3</i></td><td>1.48</td><td>1.56</td></tr><tr><td><i>MMP1</i></td><td>12.43</td><td>5.20</td></tr><tr><td><i>MMP7</i></td><td>1.44</td><td>1.82</td></tr><tr><td><i>MMP13</i></td><td>5.73</td><td>10.17</td></tr><tr><td><i>LIF</i></td><td>3.29</td><td>3.69</td></tr><tr><td><i>LGALS1</i></td><td>-0.71</td><td>-0.80</td></tr><tr><td><i>LGALS3</i></td><td>-0.59</td><td>-0.60</td></tr><tr><td><i>TGFB2</i></td><td>0.70</td><td>0.73</td></tr><tr><td><i>ACTB</i></td><td>1.09</td><td>1.04</td></tr><tr><td><i>TBP</i></td><td>1.03</td><td>1.04</td></tr><tr><td><i>IFIT1</i></td><td>1.79</td><td>10.07</td></tr><tr><td><i>IFIT3</i></td><td>2.53</td><td>13.06</td></tr><tr><td><i>MUC1</i></td><td>2.08</td><td>2.76</td></tr><tr><td><i>MUC13</i></td><td>12.55</td><td>18.09</td></tr><tr><td><i>ITGB3</i></td><td>-0.43</td><td>-0.46</td></tr><tr><td><i>ITGB6</i></td><td>1.66</td><td>1.71</td></tr><tr><td><i>CDH26</i></td><td>3.1</td><td>4.15</td></tr><tr><td><i>PKP1</i></td><td>-0.56</td><td>-0.62</td></tr></tbody></table>

R² = 0.97

https://doi.org/10.1371/journal.pone.0222081.t001
<figure><img src="image_13.png" /><figcaption>Fig 6. Changes in gene expression from RT²-qPCR at 6, 24 and 48 hours following LPS challenge. For each time point, z = (expression after LPS- expression in controls) / expression in controls) for a set of DEGs identified at 24 hours post LPS from RNA sequencing. The order of genes from left to right is organized following the amplitude of fold change at 6 hours from RT²-qPCR. NS: not significant, \* p < 0.05, \*\* p < 0.01, \*\*\* p < 0.001.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0222081.g006

Discussion

In a recent study based on the same bEEC model treated with the same concentrations of *E. coli* LPS, we previously reported, that the treatment did not alter cell viability and stimulated the proliferation of cells [9]. Despite that the stimulation of proliferation was higher with 8 µg/ mL LPS at 72 h after treatment, significant differences in gene expression at 24 hours after treatment between 2 and 8 µg/mL LPS were not observed. In addition, we found numerous changes in protein expression 72 hours after challenge indicating strong alterations of several cell functions [22]. The changes included de-regulation of proteins related to translation, metabolism, glycolysis and related oxidative stress which were over-expressed and down regulation of proteins related to cell structure and adhesion and specific proteins involved in immune-tolerance (such as GAL-1 and TGFβ). Samples from the present experiment were taken at an earlier time than for the proteomic experiment to detect changes in mRNA expression. The pathway analysis of the present study, showing differential gene expression leading to a stimulation of cell metabolism, accompanied by activation of the glycolytic and oxidative stress pathways was fully consistent with the changes observed from our proteomic analysis [22]. The down regulation of cell structure and cell adhesion pathways was also observed from both proteomics [22] and the present RNA sequencing results. When compared to other studies made in the cow [20, 21, 29, 41], human [30] and mouse [42] endometrium, our results fully confirm that a high number of differentially expressed genes relates to immune response,
response to infection and inflammation and also to cell metabolism and oxidative stress [22, 43]. Former studies in the cow, reported changes in genes involved in embryo maternal inter-action, notably interferon (IFN)-stimulated genes *RSAD2*, *MX2*, *OAS1*, *ISG15*, *BST2*; as well as several molecules involved in the intracellular recognition of pathogens or their ligands, including *IFIH1*, *MDA5*, *DEXH Asp-Glu-X-His box polypeptide 58 (DHX58)*, and up-regula-tion of a large number of chemokines such as *IL8*, *CCL4*, *CCL20*, *CXCL2*, *CXCL3*, *CSCL10*, *CXCL11*, *CCL3*, *CCL4*, *CCL11*, and *CCL20*. [20]. In addition, gene changes in *MMP1*, *MMP3*, and *MMP13* in response to LPS were also reported [44, 45].

We confirm here changes in expression for most/all these genes. However, results from this data set enlarge the list of gene expression changes corresponding to specific key molecules for embryo-maternal interaction and implantation. As cows previously infected by gram negative bacteria present infertility and high culling rate after infection [1, 4, 46] the significance of the above changes in relation with persistent inflammation and establishment of pregnancy will be discussed.

Our data indicate that epithelial cells are highly sensitive to LPS and activate strongly the corresponding inflammatory pathways. In particular, the toll-like receptor-signaling pathway was significantly affected by LPS treatment, and 19 of the over-expressed DEGs were associ-ated with this pathway including many genes encoding cytokines and chemokines. This is in full agreement with former information showing that LPS provokes the activation of the host's innate immune response by increasing TLR4- and MyD88-dependent signaling [47]and sub-sequent expression of pro-inflammatory cytokines and chemokines following binding of TLR4 [22, 47, 48], and activation of JAK/STAT signaling pathway [49] The toll-like receptor signal-ing pathway activates key molecules to drive immune-related responses towards infectious agents and attracts immune cells by the site of infection [29, 50]. In a first step, this represents one of the main defense mechanisms of the mucosal epithelium. It has also been suggested that the increased expression of cytokine and chemokine genes leads to a persistent presence of immune cells in the endometrium possibly altering implantation [50]. Chemokines can pro-mote or inhibit human trophoblast cell migration and invasion in first-trimester placenta [51]. For instance, Tumor Necrosis Factor alpha (TNF-α) has pleiotropic effects on cell growth, inflammation and innate immunity in the endometrium and is strongly involved in embryo development and implantation [10, 52, 53]. These effects can be either beneficial or detrimen-tal and high concentrations of TNF-α has been reported to be the source of implantation fail-ures and pregnancy loss [54]. Chemokines like CXCL1 and CXCL6 contribute to neutrophil recruitment and are associated with pathways involved in inflammation and apoptosis [55].

From this bEEC *in vitro* model, despite the fact that cells were exposed to relatively low dos-ages of LPS [9] and for a short time when compared to the *in vivo* situation, we observed very strong changes in expression of both cytokines and chemokines. The duration of these changes in gene expression should be evaluated further from *in vivo* studies. Such changes may alter the uterine environment and may not be favorable to the transit of spermatozoa and their sur-vival through the female genital tract [56]. However, due to the very delicate immune balance necessary for the establishment of pregnancy [4, 5] and to the very high fold change for some of the genes, it may be speculated that in cows which have been exposed to endometritis, changes in gene expression may also contribute to alter embryo-maternal relationships after infection has disappeared [57]. Undiagnosed persistent inflammation may also represent a limitation in case of embryo transfer.

Complementary to the above mechanisms related to infection and associated immune response, this study shows that many molecules specifically related to embryo development and implantation are strongly de-regulated by LPS. It has been established for a long time that in ruminants, Interferon-τ (IFNT) is the main signal for maternal recognition of pregnancy
which prevents prostaglandin F2α induced luteolysis [58]. This molecule is also critical for implantation by regulating the function of a large number of genes called IFNT-induced genes, which are regulated through the STAT-dependent signaling pathway [16, 59], which is activated at the beginning of pregnancy [60]. Activation of this pathway is driven by the developing embryo through secretion of IFNT which induces tyrosine phosphorylation of STAT-1, -2 and -3 [61]. In addition, leukemia inhibitor factor (LIF)-dependent STAT activation is critical for embryo-endometrial interaction and trophoblast invasion [10]. LIF is highly expressed in mouse uterus during receptivity phase and essential for embryo implantation [52]. Without LIF, the downstream signal transduction pathways may be crippled [62, 63]. Decreased expression or complete lack of LIF production is linked to implantation failure [10]. Contrary to what was reported above, the present experiment shows that after a short exposure of endometrial cells to LPS, both *LIF* and *STAT1* genes exhibited a strong increase in mRNA expression at 6 hours post LPS. Despite the amplitude of differential expression for these genes decreased with time, *LIF* mRNA remained strongly overexpressed by 48 hours and many downstream genes such as *MUC1*, *MUC13*, *MMP1*, *MMP7* and *MMP13* were shown to be strongly over-expressed at 48 hours. As reported above for the immune response, these results indicate that it would be useful to study the duration of these changes and their amplitude, and compare them with what is happening in response to a living embryo at the beginning of pregnancy [10]. The LPS-induced changes, if persistent, may also disturb responses of the endometrial cells to IFNT and possibly impair the success of implantation.

In addition to those referred above, our results suggest that LPS activates mechanisms altering specifically cell structure and cell adhesion properties of endometrial epithelial cells which could also be unfavorable to subsequent implantation. Successful embryo implantation requires a subtle regulation of tissue remodeling by adhesion molecules (cadherins, integrins, selectins, and MMPs) [64, 65]. The members of the cadherin superfamily mediate cell-cell interactions though calcium binding, and any possible changes could impair implantation [66]. Our results clearly show that all DEGs encoding proteins involved in cell adhesion and actin cytoskeleton which are important for the binding of growth factors to their respective receptors were down-regulated after LPS challenge. Furthermore, MMPs and its tissue inhibitors of MMPs (TIMPs) are enzymes that mediate immune response to infection, and are involved in remodeling of the extracellular matrix (ECM) during pregnancy and parturition [67]. Their activities must be well balanced for successful implantation. Decreased expression of MMP2 and TIMP3 were detected in the endometrium of women experiencing implantation failure [68, 69]. The significance of such changes should be more documented from further functional studies in the cow. However, since TIMP3 exerts a function as the major modulator of extracellular matrix degradation during implantation [70, 71], the LPS-induced down-regulation of *TIMP3* mRNA, as observed here may be unfavorable to interactions between the conceptus and the endometrium.

We also found that a group of genes coding for proteins belonging to the galectin (Gal) family was de-regulated by LPS suggesting that LPS may alter early embryo-maternal communication [72, 73]. *In vivo* and *in vitro* studies showed that Gal-1, 3 and 9 play a crucial role in the cell proliferation, adhesion processes, and modulation of innate and adaptive immunity, pro-inflammation and/or regulation of immunosuppressive activity. Gal-1 is required for establishing an immune-privileged local environment for implantation and early fetal development which relates especially from its immunosuppressive activities and key role for maternal-fetal tolerance as documented in humans and rodents [74-76]. Gal-1 is up-regulated during normal pregnancy and expressed also in human preimplantation embryos. This protein stimulates FOXP3 which controls the differentiation of T naive cells into T regulatory cells [77]. Gal-1 also positively regulates the expression of human leukocyte antigen G (HLA-G) which
inhibits NK cells and modulates cytokine secretion to control trophoblast cell invasion and to maintain a local immunotolerance during implantation [13, 14]. Probably as the result of the above, low expression in the endometrium has been associated with an increased frequency of early pregnancy failures and miscarriages [78, 79]. It has been reported that Gal-1 was present in the bovine endometrium, mainly in the lamina propria [80], but the exact roles of Gal-1 and how it regulates *BOLA* gene expression during pregnancy is not known in this species. The decreased expression of Gal-1 mRNA as observed here corroborates the decreased expression of Gal-1 protein following LPS challenge [22], which has been observed at a later stage.

In the cow, Gal-3 has been identified to be expressed in the uterus, cervix, oviduct, atretic follicles, and regressing corpus luteum [81]. Gal-3 down-regulation may be involved in the changes observed in cell adhesion as these proteins bind to a large set of molecules including integrins, laminin and fibronectin [82]. Gal-9 is expressed in all uterine cell types, including endometrial epithelia and endothelial cells [76]. Over-expression of Gal-9 associated with the increase of expression of many genes coding for pro-inflammatory cytokines suggest that Gal-9 may control positively the production of these molecules in the absence of immune cells [83].

As mentioned above for other signals, the correlation of the length of the changes in galec-tins would be useful to evaluate the duration of possible alterations in endometrial function.

In this study, the expression of the *PTHLH* gene encoding parathyroid hormone-like hormone, presented the most significantly decreased expression level following exposure to LPS (S9 Table). PTHLH has been localized in the cyclic endometrium in both the glandular and stromal compartments [84] and exerts multiple biological functions in both normal and pathological conditions. A recent study showed that significantly decreased blastocyst formation rate of Pthlh-depleted embryos, and this effect can be overturned by injection of recombinant PTHLH [85]. TGF-β-dependent signaling activates PTHLH expression by increasing transcription from the P3 promoter through a synergistic interaction of the transcription factors Smad3 and Ets1 [86], and p38 MAPK-dependent signaling controls PTHLH expression in metastatic colon cancer cells [87]. Few studies have investigated the characteristics of its expression following LPS exposure [88], however, the strong under expression observed here is very consistent with the up-stream under expression of TGF-β both contributing to impairments of cell adhesion processes.

Finally, the changes observed in genes susceptible to influence the methylation and acetylation profiles of bEECs suggest that LPS may globally induce lower methylation due to under-expression of a set of methyl transferases concomitant with effects on MBD4 which binds DNMTs to DNA. On the contrary, acetylation may be maintained through under-expression of a set of histone deacetylases and over-expression of numerous acetyl transferases. These results are consistent previous results showing that bacterial infections and viral infections induces de-methylation changes in host cells [89-91] and with the overall methylation patterns observed from Reduced Representation Bisulfite Sequencing [92].

## Conclusion

The high number of DEGs and multiple pathways affected by LPS observed in this study confirms that this molecule affects in a complex manner multiple functions of endometrial epithelial cells. Changes in proliferation, general metabolism, glycolysis, oxidative stress and immune response have been extensively described in the literature. The present study gives more insight in the way LPS affects specifically key molecules and pathways involved in embryo-maternal interactions and immuno-tolerance.

These results indicate clearly that most cellular events necessary at time of implantation for the establishment of a successful crosstalk between the endometrium and the developing
embryo are perturbed. In addition, changes in expression for genes that control methylation
may be the cause of long term consequences of previous infection on uterine function. These
findings provide new insights regarding the mechanisms by which LPS and induced inflam-
mation may alter uterine receptivity. This information showing a multiplicity of differential
gene expression patterns in response to LPS treatment suggest to perform additional studies to
evaluate further, the persistence of those changes and their functional impact on fertility.

**Supporting information**

**S1 Fig.** Overrepresented KEGG pathways in relation with immune response. A. Toll like receptor signaling pathway and B. Antigen processing and presentation pathway. Each rectangle represents a gene in the pathway. The left part of the rectangle corresponds to the comparison between control 24h and control time 0, the middle part corresponds to the comparison between LPS (2 µg/mL) and control 24h, and the right part between LPS (8 µg/mL) and control 24h. Colors indicate that the gene was either (adj P < 0.05) under-expressed (green) or over-expressed (red) whereas grey color indicates no change (adj P > 0.05).
(TIF)

**S1 Table.** Summary of RNA quality and RNA-seq reads mapping to reference genome.
(DOCX)

**S2 Table.** Distribution of the GO functional terms predicted.
(DOCX)

**S3 Table.** List of overrepresented Go terms.
(DOCX)

**S4 Table.** List of underrepresented Go terms.
(DOCX)

**S5 Table.** List of overrepresented Kegg pathways.
(DOCX)

**S6 Table.** List of underrepresented Kegg pathways.
(DOCX)

**S7 Table.** Differentially expressed genes related to diseases and responses.
(DOCX)

**S8 Table.** Top 10 most significantly over- and under-expressed DEGs associated to each responses and diseases.
(DOCX)

**S9 Table.** List of the top 100 over-expressed and under-expressed transcripts in bEEC after challenged with LPS.
(DOCX)

**Acknowledgments**

We thank the SLU Bioinformatics Infrastructure, Uppsala, Sweden for the bioinformatics
support.

**Author Contributions**

**Conceptualization:** Göran Andersson, Patrice Humblot.
**Data curation:** Tom van Schaik, Göran Andersson.

**Formal analysis:** Yongzhi Guo, Tom van Schaik, Naveed Jhamat, Adnan Niazi, Patrice Humblot.

**Investigation:** Yongzhi Guo.

**Methodology:** Yongzhi Guo, Gilles Charpigny.

**Software:** Tom van Schaik, Naveed Jhamat, Adnan Niazi.

**Supervision:** Patrice Humblot.

**Validation:** Yongzhi Guo, Patrice Humblot.

**Writing – original draft:** Yongzhi Guo, Patrice Humblot.

**Writing – review & editing:** Adnan Niazi, Metasu Chanrot, Gilles Charpigny, Jean Francois Valarcher, Erik Bongcam-Rudloff, Göran Andersson, Patrice Humblot.

## References

1. Sheldon IM, Cronin J, Goetze L, Donofrio G, Schuberth HJ. Defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle. Biology of reproduction. 2009; 81(6):1025-32. https://doi.org/10.1095/biolreprod.109.077370 PMID: 19439727; PubMed Central PMCID: PMC2784443.

2. Ordell A, Unnerstad HE, Nyman A, Gustafsson H, Bage R. A longitudinal cohort study of acute puerperal metritis cases in Swedish dairy cows. Acta veterinaria Scandinavica. 2016; 58(1):79. https://doi.org/10.1186/s13028-016-0257-9 PMID: 27832812; PubMed Central PMCID: PMC5105271.

3. Sheldon IM, Bromfield JJ. Innate immunity in the human endometrium and ovary. American journal of reproductive immunology. 2011; 66 Suppl 1:63-71. https://doi.org/10.1111/j.1600-0897.2011.01034.x PMID: 21726339.

4. Sheldon IM, Rycroft AN, Dogan B, Craven M, Bromfield JJ, Chandler A, et al. Specific strains of Escherichia coli are pathogenic for the endometrium of cattle and cause pelvic inflammatory disease in cattle and mice. PloS one. 2010; 5(2):e9192. https://doi.org/10.1371/journal.pone.0009192 PMID: 20169203; PubMed Central PMCID: PMC2820550.

5. Agrawal V JM, Jaiswal YK. LPS-Induced Implantation Failure: One of the Causes of Female Infertility. Med J Obstet Gynecol. 2013; 1(3):5.

6. Gilbert RO. The effects of endometritis on the establishment of pregnancy in cattle. Reproduction, fertility, and development. 2011; 24(1):252-7. Epub 2012/03/08. https://doi.org/10.1071/RD11915 PMID: 22394966.

7. Janeway CA Jr., Medzhitov R. Innate immune recognition. Annual review of immunology. 2002; 20:197-216. https://doi.org/10.1146/annurev.immunol.20.083001.084359 PMID: 11861602.

8. Moresco EM, LaVine D, Beutler B. Toll-like receptors. Current biology: CB. 2011; 21(13):R488-93. https://doi.org/10.1016/j.cub.2011.05.039 PMID: 21741580.

9. Chanrot M, Guo Y, Dalin AM, Persson E, Bage R, Svensson A, et al. Dose related effects of LPS on endometrial epithelial cell populations from dioestrus cows. Animal reproduction science. 2017; 177:12-24. https://doi.org/10.1016/j.anireprosci.2016.12.002 PMID: 27939589.

10. Salleh N, Giribabu N. Leukemia inhibitory factor: roles in embryo implantation and in nonhormonal traception. The Scientific World Journal. 2014; 2014:201514. Epub 2014/08/26. https://doi.org/10.1155/2014/201514 PMID: 25152902; PubMed Central PMCID: PMC4131495.

11. Robertson SA, Moldenhauer LM. Immunological determinants of implantation success. The International journal of developmental biology. 2014; 58(2-4):205-17. https://doi.org/10.1387/ijdb.140096sr PMID: 25023687.

12. Blois SM, Ilarregui JM, Tometten M, Garcia M, Orsal AS, Cordo-Russo R, et al. A pivotal role for galec tin-1 in fetomaternal tolerance. Nature medicine. 2007; 13(12):1450-7. Epub 2007/11/21. https://doi.org/10.1038/nm1680 PMID: 18026113.

13. Roussev RG, Coulam CB. HLA-G and its role in implantation (review). Journal of assisted reproduction and genetics. 2007; 24(7):288-95. https://doi.org/10.1007/s10815-007-9148-3 PMID: 17629722; PubMed Central PMCID: PMC3455004.
14. Tirado-Gonzalez I, Freitag N, Barrientos G, Shaikly V, Nagaeva O, Strand M, et al. Galectin-1 influences trophoblast immune evasion and emerges as a predictive factor for the outcome of pregnancy. Molecular human reproduction. 2013; 19(1):43-53. https://doi.org/10.1093/molehr/gas043 PMID: 23002109.

15. Mamo S, Mehta JP, McGettigan P, Fair T, Spencer TE, Bazer FW, et al. RNA sequencing reveals novel gene clusters in bovine conceptuses associated with maternal recognition of pregnancy and implantation. Biology of reproduction. 2011; 85(6):1143-51. https://doi.org/10.1095/biolreprod.111.092643 PMID: 21795669.

16. Forde N, Duffy GB, McGettigan PA, Browne JA, Mehta JP, Kelly AK, et al. Evidence for an early endometrial response to pregnancy in cattle: both dependent upon and independent of interferon tau. Physiological genomics. 2012; 44(16):799-810. https://doi.org/10.1152/physiolgenomics.00067.2012 PMID: 22759920.

17. Brooks K, Burns G, Spencer TE. Conceptus elongation in ruminants: roles of progesterone, prostaglandin, interferon tau and cortisol. Journal of animal science and biotechnology. 2014; 5(1):53. https://doi.org/10.1186/2049-1891-5-53 PMID: 25810904; PubMed Central PMCID: PMC4373033.

18. Al Moussawi K, Ghigo E, Kalinke U, Alexopoulou L, Mege JL, Desnues B. Type I interferon induction is detrimental during infection with the Whipple's disease bacterium, Tropheryma whipplei. PLoS pathogens. 2010; 6(1):e1000722. https://doi.org/10.1371/journal.ppat.1000722 PMID: 20090833; PubMed Central PMCID: PMC2798751.

19. Jaiswal M, LaRusso NF, Burgart LJ, Gores GJ. Inflammatory cytokines induce DNA damage and inhibit DNA repair in cholangiocarcinoma cells by a nitric oxide-dependent mechanism. Cancer research. 2000; 60(1):184-90. Epub 2000/01/26. PMID: 10646872.

20. Oguejiofor CF, Cheng Z, Abudureyimu A, Fouladi-Nashta AA, Wathes DC. Global transcriptomic profiling of bovine endometrial immune response in vitro. I. Effect of lipopolysaccharide on innate immunity. Biology of reproduction. 2015; 93(4):100. https://doi.org/10.1095/biolreprod.115.128868 PMID: 26353891.

21. Tesfaye D, Salilew-Wondim D, Gebremedhn S, Sohel MM, Pandey HO, Hoelker M, et al. Potential role of microRNAs in mammalian female fertility. Reproduction, fertility, and development. 2016; 29(1):8-23. https://doi.org/10.1071/RD16266 PMID: 28278789.

22. Piras C, Guo Y, Soggiu A, Chanrot M, Greco V, Urbani A, et al. Changes in protein expression profiles in bovine endometrial epithelial cells exposed to E. coli LPS challenge. Molecular bioSystems. 2017; 13 (2):392-405. https://doi.org/10.1039/c6mb00723f PMID: 28070584.

23. Ibeagha-Awemu EM, Lee JW, Ibeagha AE, Bannerman DD, Paape MJ, Zhao X. Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells. Veterinary research. 2008; 39(2):11. https://doi.org/10.1051/vetres:2007047 PMID: 18096120.

24. Zhang L, Rees MC, Bicknell R. The isolation and long-term culture of normal human endometrial epithelium and stroma. Expression of mRNAs for angiogenic polypeptides basally and on oestrogen and progesterone challenges. Journal of cell science. 1995; 108 (Pt 1):323-31. Epub 1995/01/01. PMID: 7537745.

25. Charpigny G, Reinaud P, Creminon C, Tamby JP. Correlation of increased concentration of ovine endometrial cyclooxygenase 2 with the increase in PGE2 and PGD2 in the late luteal phase. Journal of reproduction and fertility. 1999; 117(2):315-24. Epub 2000/02/26. https://doi.org/10.1530/jrf.0.1170315 PMID: 10690199.

26. Kisselbach L, Merges M, Bossie A, Boyd A. CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures. Cytotechnology. 2009; 59(1):31-44. Epub 2009/03/20. https://doi.org/10.1007/s10616-009-9190-3 PMID: 19296231; PubMed Central PMCID: PMC2677147.

27. Herath S, Lilly ST, Fischer DP, Williams EJ, Dobson H, Bryant CE, et al. Bacterial lipopolysaccharide induces an endocrine switch from prostaglandin F2alpha to prostaglandin E2 in bovine endometrium. Endocrinology. 2009; 150(4):1912-20. Epub 2008/12/06. https://doi.org/10.1210/en.2008-1379 PMID: 19056817; PubMed Central PMCID: PMC2706387.

28. Williams EJ, Fischer DP, Noakes DE, England GC, Rycroft A, Dobson H, et al. The relationship between uterine pathogen growth density and ovarian function in the postpartum dairy cow. Theriogenology. 2007; 68(4):549-59. Epub 2007/06/19. https://doi.org/10.1016/j.theriogenology.2007.04.056 PMID: 17574659; PubMed Central PMCID: PMC2702080.

29. Herath S, Fischer DP, Werling D, Williams EJ, Lilly ST, Dobson H, et al. Expression and function of Toll-like receptor 4 in the endometrial cells of the uterus. Endocrinology. 2006; 147(1):562-70. https://doi.org/10.1210/en.2005-1113 PMID: 16223858; PubMed Central PMCID: PMC2738982.

30. Hirata T, Osuga Y, Hirota Y, Koga K, Yoshino O, Harada M, et al. Evidence for the presence of toll-like receptor 4 system in the human endometrium. The Journal of clinical endocrinology and metabolism. 2005; 90(1):548-56. https://doi.org/10.1210/jc.2004-0241 PMID: 15509642.
31. Andrews S. FastQC: a Quality Control Tool for High Throughput Sequence Data [Online]. Available online at: http://wwwbioinformaticsbabrahamamacuk/projects/fastqc/. 2010.

32. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15):2114-20. Epub 2014/04/04. https://doi.org/10.1093/bioinformatics/btu170 PMID: 24695404; PubMed Central PMCID: PMC4103590.

33. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(1):15-21. Epub 2012/10/30. https://doi.org/10.1093/bioinformatics/bts635 PMID: 23104886; PubMed Central PMCID: PMC3530905.

34. Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015; 31(2):166-9. Epub 2014/09/28. https://doi.org/10.1093/bioinformatics/btu638 PMID: 25260700; PubMed Central PMCID: PMC4287950.

35. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome biology. 2014; 15(12):550. Epub 2014/12/18. https://doi.org/10.1186/s13059-014-0550-8 PMID: 25516281; PubMed Central PMCID: PMC4302049.

36. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature protocols. 2009; 4(1):44-57. https://doi.org/10.1038/nprot.2008.211 PMID: 19131956.

37. Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic acids research. 2009; 37(1):1-13. https://doi.org/10.1093/nar/gkn923 PMID: 19033363; PubMed Central PMCID: PMC2615629.

38. Huang DW, Sherman BT, Tan Q, Collins JR, Alvord WG, Roayaei J, et al. The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists. Genome biology. 2007; 8(9):R183. Epub 2007/09/06. https://doi.org/10.1186/gb-2007-8-9-r183 PMID: 17784955; PubMed Central PMCID: PMC2375021.

39. Kim YJ, Greer CB, Cecchini KR, Harris LN, Tuck DP, Kim TH. HDAC inhibitors induce transcriptional repression of high copy number genes in breast cancer through elongation blockade. Oncogene. 2013; 32(23):2828-35. https://doi.org/10.1038/onc.2013.32 PMID: 23435418; PubMed Central PMCID: PMC3676447.

40. Alger HM, Brown JM, Sawyer JK, Kelley KL, Shah R, Wilson MD, et al. Inhibition of acyl-coenzyme A: cholesterol acyltransferase 2 (ACAT2) prevents dietary cholesterol-associated steatosis by enhancing hepatic triglyceride mobilization. The Journal of biological chemistry. 2010; 285(19):14267-74. https://doi.org/10.1074/jbc.M110.118422 PMID: 20231283; PubMed Central PMCID: PMC2863169.

41. Koh YQ, Mitchell MD, Almughllig FB, Vaswani K, Peiris HN. Regulation of inflammatory mediator expression in bovine endometrial cells: effects of lipopolysaccharide, interleukin 1 beta, and tumor necrosis factor alpha. Physiological reports. 2018; 6(9):e13676. https://doi.org/10.14814/phy2.13676 PMID: 29707922; PubMed Central PMCID: PMC5925570.

42. Sheldon IM, Roberts MH. Toll-like receptor 4 mediates the response of epithelial and stromal cells to lipopolysaccharide in the endometrium. PloS one. 2010; 5(9):e12906. https://doi.org/10.1371/journal.pone.0012906 PMID: 20877575; PubMed Central PMCID: PMC2943929.

43. Batistel F, Osorio JS, Tariq MR, Li C, Caputo J, Socha MT, et al. Peripheral leukocyte and endometrium molecular biomarkers of inflammation and oxidative stress are altered in peripartal dairy cows supplemented with Zn, Mn, and Cu from amino acid complexes and Co from Co glucoheptonate. Journal of animal science and biotechnology. 2017; 8:33. https://doi.org/10.1186/s40104-017-0163-7 PMID: 28469842; PubMed Central PMCID: PMC5410708.

44. Wathes DC, Cheng Z, Fenwick MA, Fitzpatrick R, Patton J. Influence of energy balance on the somatic axis and matrix metalloproteinase expression in the endometrium of the postpartum dairy cow. Reproduction. 2011; 141(2):269-81. https://doi.org/10.1530/REP-10-0177 PMID: 21123519; PubMed Central PMCID: PMC3021913.

45. Swangchan-Uthai T, Lavender CR, Cheng Z, Fouladi-Nashta AA, Wathes DC. Time course of defense mechanisms in bovine endometrium in response to lipopolysaccharide. Biology of reproduction. 2012; 87(6):135. https://doi.org/10.1095/biolreprod.112.102376 PMID: 23077171.

46. LeBlanc SJ, Duffield TF, Leslie KE, Bateman KG, Keefe GP, Walton JS, et al. Defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows. Journal of dairy science. 2002; 85(9):2223-36. https://doi.org/10.3168/jds.S0022-0302(02)74302-6 PMID: 12362455.

47. Cronin JG, Turner ML, Goetze L, Bryant CE, Sheldon IM. Toll-like receptor 4 and MYD88-dependent signaling mechanisms of the innate immune system are essential for the response to lipopolysaccharide by epithelial and stromal cells of the bovine endometrium. Biology of reproduction. 2012; 86(2):51. https://doi.org/10.1095/biolreprod.111.092718 PMID: 22053092; PubMed Central PMCID: PMC4396703.
48. Mogensen TH. Pathogen recognition and inflammatory signaling in innate immune defenses. Clin Microbiol Rev. 2009; 22(2):240-73, Table of Contents. Epub 2009/04/16. https://doi.org/10.1128/CMR.00046-08 PMID: 19366914; PubMed Central PMCID: PMC2668232.

49. Cronin JG, Kanamarlapudi V, Thornton CA, Sheldon IM. Signal transducer and activator of transcription-3 licenses Toll-like receptor 4-dependent interleukin (IL)-6 and IL-8 production via IL-6 receptor-positive feedback in endometrial cells. Mucosal immunology. 2016; 9(5):1125-36. https://doi.org/10.1038/mi.2015.131 PMID: 26813342; PubMed Central PMCID: PMC4990777.

50. Dekel N, Gnainsky Y, Granot I, Mor G. Inflammation and implantation. American journal of reproductive immunology. 2010; 63(1):17-21. https://doi.org/10.1111/j.1600-0897.2009.00792.x PMID: 20059465; PubMed Central PMCID: PMC3025807.

51. Zhang H, Hou L, Li CM, Zhang WY. The chemokine CXCL6 restricts human trophoblast cell migration and invasion by suppressing MMP-2 activity in the first trimester. Hum Reprod. 2013; 28(9):2350-62. https://doi.org/10.1093/humrep/det258 PMID: 23814098.

52. Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F, et al. Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature. 1992; 359(6390):76-9. Epub 1992/09/03. https://doi.org/10.1038/359076a0 PMID: 1522892.

53. Granot I, Gnainsky Y, Dekel N. Endometrial inflammation and effect on implantation improvement and pregnancy outcome. Reproduction. 2012; 144(6):661-8. Epub 2012/10/03. https://doi.org/10.1530/REP-12-0217 PMID: 23028125.

54. Torchinsky A, Markert UR, Toder V. TNF-alpha-mediated stress-induced early pregnancy loss: a possible role of leukemia inhibitory factor. Chemical immunology and allergy. 2005; 89:62-71. Epub 2005/09/01. https://doi.org/10.1159/000087913 PMID: 16129953.

55. Chittur S, Parr B, Marcovici G. Inhibition of inflammatory gene expression in keratinocytes using a composition containing carnitine, thioctic Acid and saw palmetto extract. Evidence-based complementary and alternative medicine: eCAM. 2011; 2011:985345. Epub 2009/08/21. https://doi.org/10.1093/ecam/nep102 PMID: 19692448; PubMed Central PMCID: PMC3137880.

56. Novy M, Eschenbach D. & Witkin S. Infections as a Cause of Infertility. Glob libr women's med. 2008. https://doi.org/10.3843/GLOWM.10328

57. Sharkey A. Cytokines and implantation. Reviews of reproduction. 1998; 3(1):52-61. PMID: 9509989.

58. Roberts RM. Interferon-tau, a Type 1 interferon involved in maternal recognition of pregnancy. Cytokine & growth factor reviews. 2007; 18(5-6):403-8. https://doi.org/10.1016/j.cytogfr.2007.06.010 PMID: 17662642; PubMed Central PMCID: PMC2000448.

59. Spencer TE, Sandra O, Wolf E. Genes involved in conceptus-endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches. Reproduction. 2008; 135(2):165-79. https://doi.org/10.1530/REP-07-0327 PMID: 18239047.

60. Maj T, Chelmonska-Soyta A. Pleiotropy and redundancy of STAT proteins in early pregnancy. Reproduction in domestic animals = Zuchthygiene. 2007; 42(4):343-53. https://doi.org/10.1111/j.1439-0531.2006.00787.x PMID: 17635769.

61. Binelli M, Subramaniam P, Diaz T, Johnson GA, Hansen TR, Badinga L, et al. Bovine interferon-tau stimulates the Janus kinase-signal transducer and activator of transcription pathway in bovine endometrial epithelial cells. Biology of reproduction. 2001; 64(2):654-65. https://doi.org/10.1095/biolreprod64.2.654 PMID: 11159370.

62. Hu W, Feng Z, Teresky AK, Levine AJ. p53 regulates maternal reproduction through LIF. Nature. 2007; 450(7170):721-4. Epub 2007/11/30. https://doi.org/10.1038/nature05993 PMID: 18046411.

63. Lass A, Weiser W, Munafo A, Loumaye E. Leukemia inhibitory factor in human reproduction. Fertility and sterility. 2001; 76(6):1091-6. Epub 2001/12/04. https://doi.org/10.1016/s0015-0282(01)02878-3 PMID: 11730732.

64. Aplin JD. Adhesion molecules in implantation. Reviews of reproduction. 1997; 2(2):84-93. PMID: 9414470.

65. Lessey BA. Adhesion molecules and implantation. Journal of reproductive immunology. 2002; 55(1-2):101-12. PMID: 12062825.

66. Gipson IK, Blalock T, Tisdale A, Spurr-Michaud S, Allcorn S, Stavreus-Evers A, et al. MUC16 is lost from the uterodome (pinopode) surface of the receptive human endometrium: in vitro evidence that MUC16 is a barrier to trophoblast adherence. Biology of reproduction. 2008; 78(1):134-42. https://doi.org/10.1095/biolreprod.106.058347 PMID: 17942799.

67. Tency I, Verstraelen H, Kroes I, Holtappels G, Verhasselt B, Vaneechoutte M, et al. Imbalances between matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in maternal serum during preterm labor. PloS one. 2012; 7(11):e49042. https://doi.org/10.1371/journal.pone.0049042 PMID: 23145060; PubMed Central PMCID: PMC3493509.
68. Konac E, Alp E, Onen HI, Korucuoglu U, Biri AA, Menevse S. Endometrial mRNA expression of matrix metalloproteinases, their tissue inhibitors and cell adhesion molecules in unexplained infertility and implantation failure patients. Reproductive biomedicine online. 2009; 19(3):391-7. Epub 2009/09/26. PMID: 19778485.

69. Salamonsen LA, Zhang J, Hampton A, Lathbury L. Regulation of matrix metalloproteinases in human endometrium. Hum Reprod. 2000; 15 Suppl 3:112-9. Epub 2000/10/21. https://doi.org/10.1093/humrep/15.suppl_3.112 PMID: 11041227.

70. Hurst PR, Palmay RD. Matrix metalloproteinases and their endogenous inhibitors during the implantation period in the rat uterus. Reproduction, fertility, and development. 1999; 11(7-8):395-402. Epub 2000/12/02. PMID: 11101274.

71. Whiteside EJ, Jackson MM, Herington AC, Edwards DR, Harvey MB. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 are key regulators of extracellular matrix degradation by mouse embryos. Biology of reproduction. 2001; 64(5):1331-7. Epub 2001/04/25. https://doi.org/10.1095/biolreprod64.5.1331 PMID: 11319136.

72. Popovici RM, Krause MS, Germeyer A, Strowitzki T, von Wolff M. Galectin-9: a new endometrial epithelial marker for the mid- and late-secretory and decidual phases in humans. The Journal of clinical endocrinology and metabolism. 2005; 90(11):6170-6. Epub 2005/08/18. https://doi.org/10.1210/jc.2004-2529 PMID: 16105962.

73. Than NG, Romero R, Balogh A, Karpati E, Mastrolia SA, Staretz-Chacham O, et al. Galectins: Double-edged Swords in the Cross-roads of Pregnancy Complications and Female Reproductive Tract Inflammation and Neoplasia. Journal of pathology and translational medicine. 2015; 49(3):181-208. https://doi.org/10.4132/jptm.2015.02.25 PMID: 26018511; PubMed Central PMCID: PMC4440931.

74. Gomez-Chavez F, Castro-Leyva V, Espejel-Nunez A, Zamora-Mendoza RG, Rosas-Vargas H, Cincino-Diaz JC, et al. Galectin-1 reduced the effect of LPS on the IL-6 production in decidual cells by inhibiting LPS on the stimulation of IkappaBzeta. Journal of reproductive immunology. 2015; 112:46-52. Epub 2015/08/01. https://doi.org/10.1016/j.jri.2015.07.002 PMID: 26226212.

75. Heusschen R, Griffioen AW, Thijssen VL. Galectin-9 in tumor biology: a jack of multiple trades. Biochimica et biophysica acta. 2013; 1836(1):177-85. https://doi.org/10.1016/j.bbcan.2013.04.006 PMID: 23648450.

76. Shimizu Y, Kabir-Salmani M, Azadbakht M, Sugihara K, Sakai K, Iwashita M. Expression and localization of galectin-9 in the human uterodome. Endocrine journal. 2008; 55(5):879-87. PMID: 18506087.

77. Cedeno-Laurent F, Dimitroff CJ. Galectin-1 research in T cell immunity: past, present and future. Clin Immunol. 2012; 142(2):107-16. https://doi.org/10.1016/j.clim.2011.09.011 PMID: 22019770; PubMed Central PMCID: PMC3266984.

78. Barrientos G, Freitag N, Tirado-Gonzalez I, Unverdorben L, Jeschke U, Thijssen VL, et al. Involvement of galectin-1 in reproduction: past, present and future. Human reproduction update. 2014; 20(2):175-93. https://doi.org/10.1093/humupd/dmt040 PMID: 24077937.

79. Jeschke U, Toth B, Scholz C, Friese K, Makrigiannakis A. Glycoprotein and carbohydrate binding protein expression in the placenta in early pregnancy loss. Journal of reproductive immunology. 2010; 85 (1):99-105. https://doi.org/10.1016/j.jri.2009.10.012 PMID: 20299109.

80. Froehlich R, Hambruch N, Haeger JD, Dilly M, Kaltner H, Gabius HJ, et al. Galectin fingerprinting detects differences in expression profiles between bovine endometrium and placentomes as well as early and late gestational stages. Placenta. 2012; 33(3):195-201. https://doi.org/10.1016/j.placenta.2011.12.016 PMID: 22239761.

81. Kim M, Kim S, Kim H, Joo HG, Shin T. Immunohistochemical localization of galectin-3 in the reproductive organs of the cow. Acta histochemica. 2008; 110(6):473-80. Epub 2008/06/11. https://doi.org/10.1016/j.acthis.2008.02.004 PMID: 18541290.

82. Margadant C, van den Bout I, van Boxtel AL, Thijssen VL, Sonnenberg A. Epigenetic regulation of galectin-3 expression by beta1 integrins promotes cell adhesion and migration. The Journal of biological chemistry. 2012; 287(53):44684-93. https://doi.org/10.1074/jbc.M112.426445 PMID: 23118221; PubMed Central PMCID: PMC3531783.

83. Norling LV, Perretti M, Cooper D. Endogenous galectins and the control of the host inflammatory response. The Journal of endocrinology. 2009; 201(2):169-84. https://doi.org/10.1677/JOE-08-0512 PMID: 19155286.

84. Hoshi S, Morimoto T, Saito H, Ichizuka K, Matsuoka R, Yanaihara A, et al. PTHrP and PTH/PTHrP receptor expressions in human endometrium. Endocrine journal. 2001; 48(2):219-25. Epub 2001/07/18. PMID: 11456271.

85. Guo L, Qi ST, Miao DQ, Liang XW, Li H, Ou XH, et al. The roles of parathyroid hormone-like hormone during mouse preimplantation embryonic development. PloS one. 2012; 7(7):e40528. Epub 2012/07/01.
19. https://doi.org/10.1371/journal.pone.0040528 PMID: 22808183; PubMed Central PMCID: PMC3396650.

86. Lindemann RK, Ballschmieter P, Nordheim A, Dittmer J. Transforming growth factor beta regulates parathyroid hormone-related protein expression in MDA-MB-231 breast cancer cells through a novel Smad/Ets synergism. The Journal of biological chemistry. 2001; 276(49):46661-70. Epub 2001/10/09. https://doi.org/10.1074/jbc.M105816200 PMID: 11590145.

87. Urosevic J, Garcia-Albeniz X, Planet E, Real S, Cespedes MV, Guiu M, et al. Colon cancer cells colonize the lung from established liver metastases through p38 MAPK signalling and PTHLH. Nature cell biology. 2014; 16(7):685-94. Epub 2014/06/02. https://doi.org/10.1038/ncb2977 PMID: 24880666.

88. Salilew-Wondim D, Ibrahim S, Gebremedhn S, Tesfaye D, Heppelmann M, Bollwein H, et al. Clinical and subclinical endometritis induced alterations in bovine endometrial transcriptome and miRNome profile. BMC genomics. 2016; 17(1):218. Epub 2016/03/12. https://doi.org/10.1186/s12864-016-2513-9 PMID: 26965375; PubMed Central PMCID: PMC4785637.

89. Macnab JC, Adams RL, Rinaldi A, Orr A, Clark L. Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus. Molecular and cellular biology. 1988; 8(4):1443-8. https://doi.org/10.1128/mcb.8.4.1443 PMID: 2837642; PubMed Central PMCID: PMC363301.

90. Pacis A, Tailleux L, Morin AM, Lambourne J, Maclsaac JL, Yotova V, et al. Bacterial infection remodels the DNA methylation landscape of human dendritic cells. Genome research. 2015; 25(12):1801-11. https://doi.org/10.1101/gr.192005.115 PMID: 26392366; PubMed Central PMCID: PMC4665002.

91. Engdahl E, Dunn N, Niehusmann P, Wideman S, Wipfler P, Becker AJ, et al. Human Herpesvirus 6B Induces Hypomethylation on Chromosome 17p13.3, Correlating with Increased Gene Expression and Virus Integration. Journal of virology. 2017; 91(11). https://doi.org/10.1128/JVI.02105-16 PMID: 28298607; PubMed Central PMCID: PMC5432873.

92. Jhamat N, Guo Y, Niazi A, Ivanova E, Kelsey G, Bongcam-Rudloff E, et al. Identification of differentially methylated regions in the genome of bovine endometrial epithelial cells (BEEC) challenged by E. coli LPS and its effect on transcription. In: Proceedings of the COST Epiconcept Conference, 2016, Giardini-Naxos, Italy Milan: University of Milan. 2016; CoST. Abstr. 32:1.