Thin endometrium transcriptome analysis reveals a potential mechanism of implantation failure

Abstract Aim Although a thin endometrium has been well recognized as a critical factor in implantation failure, little information is available regarding the molecular mechanisms. The present study investigated these mechanisms by using genome‐wide mRNA expression analysis. Methods Thin and normal endometrial tissue was obtained from a total of six women during the mid‐luteal phase of the menstrual cycle. The transcriptomes were analyzed with a microarray. Differentially expressed genes were classified according to Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Results The study identified 318 up‐regulated genes and 322 down‐regulated genes in the thin endometrium, compared to the control endometrium. The GO and KEGG pathway analyses indicated that the thin endometrium possessed aberrantly activated immunity and natural killer cell cytotoxicity that was accompanied by an increased number of inflammatory cytokines, such as IFN‐γ. Various genes that were related to metabolism and anti‐oxidative stress were down‐regulated in the thin endometrium. Conclusion Implantation failure in the thin endometrium appears to be associated with an aberrantly activated inflammatory environment and aberrantly decreased response to oxidative stress.


| Transcriptome analysis
The total RNAs were isolated from the tissues by using TRIzol reagent Control, normal-thickness endometrium; LMP, last menstrual period; thin, thin endometrium.
for the hierarchical clustering analysis and principal component analysis (PCA). Those genes whose expressions in the thin and normal endometrium differed by at least a factor of 2 and that had a false discovery rate of <.05 were judged as showing a significant difference.

| Bioinformatics
A hierarchical clustering and a PCA were conducted in R v. 3.2.4. 11 DAVID Bioinformatics Resources v. 6.7 (http://david.abcc.ncifcrf. gov/home.jsp) was used to determine whether the functional annotation of the differentially expressed genes was enriched for specific Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. 12 Statistical significance was assessed with a modified Fisher's exact test. In the GO and KEGG analyses, P<.01 and P<.05, respectively, were considered to indicate significant enrichment. All the information from the GO and KEGG pathway analyses is shown in Tables S1-S4.

| Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses for the downregulated genes in the thin endometrium
The down-regulated genes in the thin endometrium were related to metabolic processes, such as "small molecule catabolic process," "single-organism catabolic process," "organic acid catabolic process," and "carboxylic acid catabolic process" (Tables 4 and S3).

| Up-regulated genes in the thin endometrium
Although a thin endometrium is known to be involved in implantation failure, the mechanism has not been elucidated. The authors recently found that a high level of blood flow impedance of the uterine radial artery underlies a thin endometrium. 4 The present study investigated

| Down-regulated genes in the thin endometrium
The GO analyses indicated that the down-regulated genes included a number of genes related to catabolic processes, which are essential in breaking down large molecules, such as polysaccharides, lipids, and proteins, into smaller units, such as monosaccharides, fatty acids, and amino acids. These small units are used to synthesize acetyl-CoA, which is needed to produce adenosine 5′-triphosphate in the citrate cycle. Acetyl-CoA is also used for the synthesis of ketone bodies, which can be an energy source. The KEGG pathway analysis showed that the genes related to butanoate metabolism were downregulated in the thin endometrium. Genes, such as CPT1, HMGCS2, and OXCT1, are essential for generating acetyl-CoA and ketone bodies in butanoate metabolism. [13][14][15] Butanoate is a substrate that is used to generate energy in both aerobic and anaerobic processes. The present findings suggest that energy synthesis in the cell is impaired in the thin endometrium. The deficiency of energy could be associated with cellular dysfunction in the endometrium, resulting in implantation failure.
In conclusion, the present study revealed that the thin endometrium possesses an aberrant Th1-pro-inflammatory/Th2-antiinflammatory balance and increased cytotoxic condition and that a protective response to oxidative stress is impaired. These aberrant molecular mechanisms in the thin endometrium might be associated with implantation failure. These findings could lead to better treatments for patients with implantation failure as a result of a thin endometrium. T A B L E 5 Kyoto Encyclopedia of Genes and Genomes pathway analysis for the genes that were down-regulated in the thin endometrium