MicroRNA Profiles in Spontaneous Decidualized Menstrual Endometrium and Early Pregnancy Decidua with Successfully Implanted Embryos.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression and play fundamental roles in many biological processes, including cell differentiation and proliferation. In general, miRNAs inhibit translation or induce mRNA degradation by binding to the 3’ untranslated region of the target mRNA. To date, over 2,000 human miRNAs have been registered in miRBase v19.0 [ 1 , 2 ]. Abnormal miRNA expression is associated with a number of benign gynecologic conditions, malignancies, and fertility disorders [ 3 ]. The abundance of specific miRNAs as well as relative differences in expression level can be assessed through miRNA sequencing. Several independent studies have profiled miRNA expression in secretory and proliferative endometria [ 4 – 6 ]. Other studies have shown that differences in miRNA expression occur during the implantation window in the secretory endometrium of patients either with or without endometriosis [ 7 , 8 ]. However, to date, no reports have described the miRNA profiles of the spontaneous decidualized menstrual endometrium and early pregnancy decidua. Menstruation is triggered by a decline in the level of circulating progesterone and involves the periodic discharge of blood and mucosal tissue, which consists of the decidualizing superficial endometrium. In response to declining progesterone levels, spontaneous decidualization leads to menstrual shedding and subsequent regeneration of the endometrium. In contrast to the case with most mammals, embryo implantation is not required for decidualization of the human endometrium. Furthermore, menstruation and cyclic regeneration of the endometrium involve recruitment of stem cells, imparting an intrinsic capacity for adaptation of the decidual response to maximize reproductive success. Once initiated, decidualization proceeds through distinct phenotypic phases involving endometrial receptivity, embryo selection, and ultimately, resolution of pregnancy at pregnancy initiation [ 9 , 10 ]. Based on our previous study [ 11 ], we hypothesized that various miRNAs might play a role in embryo implantation and decidualization. However, the profile of miRNA expression in the menstrual endometrium remains to be elucidated [ 12 , 13 ]. Characterization of the miRNomes of the human early pregnancy decidua and menstrual endometrium via deep sequencing might thus identify those decidualization-associated miRNAs that are involved in embryo implantation [ 14 , 15 ]. The aim of the present study, therefore, was to identify miRNAs that play an important role in embryo implantation by characterizing the expression of miRNAs in the human menstrual endometrium and early pregnancy decidua. The miRNA profile of the early pregnancy decidua was compared with that of the menstrual phase in order to obtain new insights into the roles of miRNAs at conception.

A total of 2,042 known miRNAs were expressed in the human decidua and endometrium samples analyzed. In addition, 177 miRNAs were differentially expressed between groups, including 88 that were up-regulated and 89 that were down-regulated ( S2 Table ). Clean sequence reads for the abundantly expressed miRNAs (>10,000 transcripts per million [TPM]) were used for further analyses. The known miRNAs in both groups exhibited a similar distribution in each library, based on classification by TPM. In normal decidua, 96.82% of miRNAs were expressed at low levels (10,000 TPM). However, the abundantly expressed miRNAs accounted for 87.11% of all miRNA reads. In the terminated-pregnancy decidua, 97.01% of miRNAs were expressed at 10,000 TPM. The abundantly expressed miRNAs accounted for 87.6% of all miRNA reads. The three most abundantly expressed miRNAs in both groups were hsa-let-7f-5p, hsa-miR-199a-3p, and hsa-miR-199b-3p, accounting for 73.28%, 6.91%, and 6.91% of the miRNome, respectively. The differential expression of six abundantly expressed miRNAs (miR-34c, miR-92a, miR-181a-5p, miR-191, miR-10a-5p, and let-7f-5p) was verified by qRT-PCR analysis of the 35 normal-pregnancy deciduas and 25 menstrual endometria ( Fig 1 ). In addition, qPCR analysis confirmed the differential expression of four significantly down-regulated miRNAs and two significantly up-regulated miRNAs in the normal-pregnancy deciduas examined. The miRNA expression level was normalized to that of U6 snRNA. “*” represents P value <0.05, “**” represents P value <0.01, and “***” represents P value <0.001. All experiments were repeated with 3 replicates. A total of 62 novel miRNAs were some identified in deciduas and some in the menstrual endometria ( Table 1 ). The expression of four novel miRNAs (miR-1, miR-12, miR-18, and miR-110) was markedly altered in the decidua, but these miRNAs were expressed at low levels (data not shown). Candidates with sequence overlap with known miRNAs (but having a distinct mature miRNA sequence: isomiRs) and sequence alignments of novel miRNA candidates with known miRNAs of other species are shown. A total of 37,513 target genes were predicted using Mireap software. The majority of the target genes were found to be involved in cellular processes, regulation of biological processes, and metabolic processes. The pathways with the most genes targeted by the identified miRNAs as determined by KEGG analysis are shown in Fig 2 . Horizontal axis indicates the annotated signaling pathways identified, and the vertical axis indicates the number of genes in each pathway to which miRNAs were predicted to bind. A number of candidate miRNA-target genes involved in establishing and maintaining pregnancy were identified in previous studies [ 11 , 23 , 24 ]. These binding-site predictions from our analyses suggest that interactions between miRNAs and mRNAs are involved in the maintenance of early pregnancy ( Fig 3 ). (a) Let-7f-5p was predicted to bind to a site on the IGF2BP-1 mRNA. (b) Let-7g-5p was predicted to bind to a site on the IGF-2R mRNA. To further investigate the effect of let-7 miRNA on decidualization, the expression of target genes was evaluated following overexpression or knockdown of the miRNAs mediated by corresponding miRNA mimics or inhibitors, respectively. Because miRNAs of the let-7 family were found to be abundantly expressed in decidua in a previous study [ 11 ], our in vitro experiments had focused solely on the effects of let-7f and let-7g-5p. In the present study, we found that the expression of IGF2BP-1 mRNA increased in HEK293T and hESCs cells transfected with let-7f-5p inhibitor and decreased in cells transfected with let-7f-5p mimic ( Fig 4 ). Likewise, the expression of IGF2R mRNA increased in cells transfected with let-7g-5p inhibitor and decreased in cells transfected with let-7g-5p mimic ( Fig 5 ). (a) Overexpression of let-7f-5p in HEK293T and hESCs cells. Cells were cultured and incubated with hsa-let-7f-5p mimic or negative control. Levels of let-7f-5p were determined using qPCR. (b) Knockdown of let-7f miRNA by transfection with hsa-let-7f-5p inhibitor. Cells were cultured and incubated with hsa-let-7f-5p inhibitor or negative control. Levels of let-7f-5p were determined using qPCR. (c-d) Expression of IGF2BP-1 mRNA, as determined using qPCR analysis. Experiments were replicated three times, and error bars represent standard error. **P<0.01, ***P<0.001. (a) Overexpression of let-7g-5p in HEK293T and hESCs cells. Cells were cultured and incubated with let-7g-5p mimic or negative control. Levels of let-7g-5p were determined using qPCR. (b) Knockdown of let-7g-5p miRNA by transfection with hsa-let-7g-5p inhibitor. Cells were cultured and incubated with hsa-let-7g-5p inhibitor or negative control. Levels of let-7g-5p were determined using qPCR. (c-d) Expression of IGF2R mRNA, as determined using qPCR analysis. Experiments were replicated three times, and error bars represent standard error. *P<0.05, **P<0.01 ***P<0.001.

In summary, this study characterized the miRNomes of the human decidua and menstrual endometrium during pregnancy, providing new data that should prove useful in subsequent investigations of the roles played by miRNAs during decidualization from a maternal aspect. Our in vitro data reveal an inverse relationship between the expression of the abundant let-7 miRNAs and their target genes, suggesting that miRNAs are involved in mediating endometrial receptivity and decidualization.

Endometrial biopsies were performed on the second day of the menstrual cycle in 25 women who suffered from secondary infertility due to either tubal or male factors and who received treatment between October 2011 and April 2012 in the Reproductive Center of Inner Mongolia University Affiliated Hospital (Inner Mongolia, China). Participants ranged in age from 22 to 39 years (mean, 31 years). In addition, decidua tissue was collected from 35 women (mean age, 28.6 years; range, 19–39 years) with a normal pregnancy (based on ultrasound evidence of fetal heart activity) who underwent pregnancy termination at 6–8 weeks of gestation. Samples were collected for miRNA and associated target gene analysis. The decidua was separated from the trophoblast, and each tissue sample was immediately frozen in liquid nitrogen. Six normal-pregnancy deciduas and six menstrual endometria were collected for sequencing analysis from as previous mentioned. The women in each group from whom specimens were collected for sequencing did not differ significantly in age. The study was approved by the Institute Research Ethics Committee of the Inner Mongolia Medical University Affiliated Hospital, and written informed consent was obtained from all participants. Total RNA (including miRNAs) was extracted using RNAiso plus (Takara, Shiga, Japan). All samples passed the RNA quality control criteria for Solexa sequencing (RNA 28S:18S ≥1.5, RNA integrity number ≥8). The sequencing procedure was as described previously [ 11 ]. Small RNAs perfectly matching the miRBase precursor sequence and those identified as known miRNA editing polymorphisms in the miRBase 19.0 database [ 16 , 17 ] were annotated and calculated. Mireap software [ 18 ] was used to predict novel miRNAs, as described previously [ 11 ]. The function of an miRNA is ultimately determined by the genes that the miRNA targets and by the effect of the miRNA on the expression of those genes. The RNAhybrid software [ 19 ] was used to predict the target genes of the novel miRNAs identified in this study [ 20 , 21 ]. Human embryonic kidney (HEK293T) cells and human endometrial stromal cells (hESCs) were obtained from the American Type Culture Collection and cultured in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco). Let-7f-5p and let-7g-5p mimics (dsRNA oligonucleotides) and inhibitors (single-stranded chemically modified oligonucleotides) (GenePharma, Shanghai, China) were used for the in vitro overexpression and inhibition of let-7f-5p and let-7g-5p activity, respectively in S1 Table . Cells were plated in 6-well plates and cultured to 50–70% confluence prior to transfection. Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) was used for transfection according to the manufacturer’s instructions. Cells were transfected with RNAs at a final concentration of 10 nM; negative controls for the miRNA mimics and inhibitors were transfected as matched controls. Total RNA (1 μg) from each sample was used for the reverse transcriptase (RT) reaction to generate cDNAs using a microRNA RT kit (Takara). Quantitative real-time PCR was performed (in triplicate) using a SYBR Green PCR kit (Takara) according to the manufacturer’s instructions with an ABI Prism 7300HT real-time PCR system (Thermo Fisher Scientific, MA, USA). The PCR primers used in the study are listed in S1 Table . The expression levels of miRNAs and the predicted target genes ( IGF2BP-1 and IGF2R ) were normalized to those of the U6 snRNA and ACTB , respectively. Relative expression was calculated using the 2 -ΔΔCT method [ 22 ]. All data are presented as the mean ± SD. The statistical significance of differences between experimental groups was assessed using the Student’s t-test or nonparametric test, and a P-value <0.05 was considered indicative of statistical significance.

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