Abstract
Study question
What is the qualitative and quantitative profile of microRNAs (miR) and their sequence variants - isomiRs, and how it changes during the menstrual-cycle phase transitions?
Summary answer
Time-course analysis of endometrial miR/isomiR profiles has shown that menstrual-phase transitions cause widespread and complex changes in miR gene expression and processing.
What is known already
Embryo implantation depends on the receptivity of the endometrium during the window of implantation, when ovarian hormones and genetic factors coordinate the development of the uterine lining and prepare it for embryo implantation. The most important factors for successful implantation studied so far are the embryo itself, the histological dating of the endometrium and its molecular genetic characteristics, including miRs. With the rapid development of next-generation sequencing technologies, it has become clear that miR genes have the potential to produce not only miR but also variants (isomiRs) thereof, which can differ in sequence and length and can be functionally significant.
Study design, size, duration
miR/isomiR landscape was assessed by small RNA sequencing of endometrial biopsy samples at 4 time points of endometrial cycle covering the proliferative and secretory phases. Healthy, fertile, female volunteers took part in the study lasting one and a half years. For accurate phase dating, human chorionic gonadotropin (hCG) was administrated, and ultrasonic, histological and hormonal assessments were done at each time point. Statistically significant data of miR/isomiR identification and expression dynamics was considered for analysis.
Participants/materials, setting, methods
Participant choice criteria - at least one child born, problem-free pregnancies, no diseases or allergies; hCG application time determined according follicle and endometrium ultrasound scanning, and ovarian hormone levels; endometrial biopsies taken at hGC (before hormone application), hGC+2, hGC+7, hGC+9 time points; small RNAseq completed by Karolinska Institute, Sweden; miR/isomiR identified using local Galaxy instance with an in-built workflow and tools developed by our laboratory; differential expression and target prediction evaluated with DESeq2 and miRDB,resp.
Main results and the role of chance
Within the cohort of patients, across the four study time points, the small RNAseq data revealed numbers of miRs and isomiRs to be changed. The largest statistically significant changes in their expression were found at LH + 9. The miR families that showed the largest number of members with altered expression were miR125a, miR30d, miR449c, miR92a/b and miR99a. The expression levels tended to decrease in the miR125a and miR92a families and to increase in the miR10a and miR449c families during the three studied time points of the cycle compared to the proliferative phase. Among those affected, the number of isomiRs, including templated and non-templated isomiRs, was much higher than that of miRs. For example, the ratio of the significantly altered miRs/templated isomiRs/non-templated isomiRs was 6/16/11 at LH + 9. Templated isomiRs of hsa-miR–148a–3p, hsa-miR–30d–5p and hsa-miR–449c–5p were among the most upregulated, while several templated and non-templated isomiRs of hsa-miR–125–5p were the most downregulated at LH + 9. Of particular interest are those isomiRs, in which the seed site is shifted compared to the reference miRs and results in altered target transcripts. Target prediction of the most affected isomiR of hsa-miR–449c–5p identified new targets of target scores much higher than of the reference miR.
Limitations, reasons for caution
IsomiRs are a source of novel biomarkers for clinical diagnosis. An important next step is the validation of the in-silico predicted miRs/isomiRs and their target transcripts by RT-qPCR in larger number of individuals. Expression profiles should be associated with the dominant cell type in the endometrial biopsy preparation.
Wider implications of the findings: MiR/isomiR signatures, together with those of their target mRNAs, can be applied to distinguish the endometrial phases, especially the implantation window, as well as for diagnosing endometrial dysfunction. It is worth investigating the possibility of miRs/isomiRs being used as biomarkers not only in endometrial biopsy but also in liquid biopsy.
Trial registration number
The Bulgarian National Science Fund КП–06 Н31/2
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