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A Comprehensive Two-Centre RNA-Seq Study Reveals Changes in Endometrial and Blood miRNome at Mid-Secretory Phase in Fertile Women and in Patients with Recurrent Implantation Failure

| Reproductive Health
Authors:
*Agne Velthut-Meikas,1 Signe Altmäe,2 Kadri Rekker,3 Marina Suhorutšenko,3 Maire Peters,3 Juan F. Martinez-Blanch,4 Francisco M. Codoñer,5 Felipe Vilella,5 Carlos Simón,6 Andres Salu-mets3
Disclosure:

The authors have declared no conflicts of interest.

Acknowledgements:

This study was funded by the Estonian Ministry of Education and Research (Grant IUT34-16); Enterprise Estonia (Grant EU48695); the EU-FP7 Eurostars program (Grant NOTED, EU41564); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, Grant SARM, EU324509); Horizon 2020 innovation programme (WIDENLIFE, 692065); a grant from the University of Granada (Incorporación de jóvenes doctores); and a grant from the Spanish Ministry of Economy, Industry and Competitiveness–MINECO–(RYC-2016-21199 and Grant ENDORE SAF2017-87526).

Citation
EMJ Repro Health. ;4[1]:63-64. Abstract Review No. AR4.

Each article is made available under the terms of the .

The molecular changes in the endometrium that are involved in the establishment of the window of embryo implantation (WOI) are of interest for determining the reasons for recurrent implantation failure (RIF) in some in vitro fertilisation (IVF) patients. Genome-wide gene expression studies performed on endometrial samples, however, have several limitations: firstly, the study groups are small and the results from one research centre are poorly reproducible by another;1 secondly, high-throughput sequencing studies on gene expression regulation by microRNA have only recently started to emerge, and also include only small sample numbers.2,3 There has been no indication of whether molecular markers revealing optimal WOI time could also be identified from blood.

To address these concerns, the authors collected endometrial and blood samples from two independent research centres, one from Estonia (EST) and one from Spain (ESP). All study participants performed urinary ovulation tests (LH-tests). Altogether, 39 fertile volunteers (women with a history of at least one live birth) donated samples twice: 2 and 8 days after obtaining a positive LH-test (LH+2 and LH+8 timepoints, respectively). In addition, 38 RIF patients (women with a history of ≥3 unsuccessful IVF procedures involving embryo transfer) donated their samples at LH+8. All samples underwent genome-wide mRNA and small RNA deep sequencing (Illumina Inc., San Diego, California, USA). Results were compared between LH+2 versus LH+8 timepoints in the group of fertile women and between fertile versus RIF women at LH+8. MicroRNA with a statistically significant change in expression levels were further passed into downstream gene ontology analysis, during which potential microRNA target genes were sought from differentially expressed mRNA from the same samples. Only consistent results between EST and ESP centres are reported.

The authors observed that the expression of 91 microRNA changed in the endometrium of fertile women during the establishment of WOI when LH+2 and LH+8 timepoints were compared. These microRNA are involved in processes like glucocorticoid receptor, oestrogen receptor, and growth hormone receptor signalling, among others. In addition to already known microRNA, a novel microRNA sequence was identified, the expression of which was increased 37 times in LH+8 samples compared to LH+2 samples. Bioinformatic target prediction algorithms suggest that this microRNA is involved in regulating cell cycle progression.

No differences in microRNA levels were observed when blood samples were compared between LH+2 and LH+8 samples from fertile women. A similar result has been previously demonstrated for blood plasma microRNA, confirming that endometrial cyclic changes do not reflect in systemic miRNome.4

When the endometrial samples from fertile women were compared to RIF patients, 21 microRNA showed significantly different expression levels. These microRNA are predicted to be regulators of the STAT3 and CDK5 signalling pathways. In addition, the level of hsa-miR-30a-5p was significantly higher in the blood samples of RIF patients. However, the molecular background of this finding still needs to be elucidated.

In conclusion, our study confirms several known microRNA, reveals novel candidates as molecular markers for WOI, and sheds light on cellular processes that are perturbed in the endometria of RIF patients. Since the reported findings are based on larger study groups and two independent population cohorts, we expect that these results can be successfully replicated by other centres. The use of these markers in the clinical setting, however, still needs to be validated.

References
Altmäe S et al. Meta-signature of human endometrial receptivity: A meta-analysis and validation study of transcriptomic biomarkers. Sci Rep. 2017;7(1):10077. Sha AG et al. Genome-wide identification of micro-ribonucleic acids associated with human endometrial receptivity in natural and stimulated cycles by deep sequencing. Fertil Steril. 2011;96(1):150-5. Sigurgeirsson B et al. Comprehensive RNA sequencing of healthy human endometrium at two time points of the menstrual cycle. Biol Reprod. 2017;96(1):24-33. Rekker K et al. Circulating microRNA profile throughout the menstrual cycle. PLoS One. 2013;8(11):e81166.