Introduction
Endometriosis is a common oestrogen-related gynaecological disorder that is known to cause serious pelvic pain and infertility and affects 6–10% of reproductive-aged women and 20–50% of infertile women [
1,
2]. Previous studies have reported that the quantity and quality of retrieved oocytes are decreased in women with endometriosis, resulting in decreased fertilization rates and poor early embryogenesis during in vitro fertilization (IVF) and embryo transfer [
3,
4].
MicroRNAs (miRNAs) are highly conserved, single-stranded, non-coding RNA molecules comprising 20–24 nucleotides. They regulate gene expression, primarily at the posttranscriptional level, through various mechanisms, with positive or negative effects [
5,
6]. Several studies have identified the presence of miRNAs in the follicular fluid (FF) of humans [
7]. The FF provides a very specialized microenvironment and contains various hormones, proteins, metabolites, and regulatory molecules that play critical roles in oocyte quality and maturation. Recent studies have reported that the miRNA expression profiles of human FF can be used to distinguish high-quality embryos from low-quality ones [
8] and affect pathways of ovarian function and follicle development [
9]. In addition, we previously found that some miRNAs in the follicular fluid were associated with viable blastocyst formation [
10].
However, to date, no studies have reported the miRNA expression profile of the FF of women with endometriosis. Understanding the regulation of miRNAs in the FF and identifying their specific targets and functions could offer novel insights into the aetiology of endometriosis and the associations between endometriosis and abnormal oocytes and embryo development.
The aim of this study was to investigate the miRNA expression profile of the FF of women with endometriosis relative to that of women with male factor infertility. We utilized quantitative reverse transcription polymerase chain reaction (qRT-PCR) to identify the differential expression of miRNAs associated with endometriosis. In addition, we investigated the effects and molecular mechanisms of these miRNAs in oocytes and embryonic development potential by injecting the corresponding inhibitor oligonucleotides into human and mouse oocytes.
Discussion
In the present study, the clinical data indicated that the number of oocytes, fertilization rate, and number of available embryos were significantly reduced in endometriosis patients relative to non-endometriosis patients. The TaqMan miRNA arrays and qRT-PCR validated the finding that hsa-miR-451 showed significantly lower expression in the endometriosis group than in the control group. Subsequently, our in vitro study suggested that miR-451 significantly affected embryo development potential. Functional analysis of mouse/human oocytes and embryos showed that the aberrant expression of WNT signalling pathway genes might negatively affect oocyte competence for fertilization and pre-implantation embryo development. To the best of our knowledge, this is the first quantitative assessment of FF miRNAs that could explain aberrant oocyte and embryo development in women with endometriosis.
FF is a complex mixture of proteins, hormones, vitamins, cytokines, and metabolites, and multiple studies have indicated that the constituents of FF may affect the acquisition of oocyte competence in women with endometriosis [
16]. The presence of miRNAs in human FF was first described by Sang et al. (2013) [
7]. They demonstrated that some miRNAs could regulate steroidogenesis in vitro and showed that their expression levels were correlated with polycystic ovary syndrome (PCOS) in vivo. Recently, it was reported that miRNA levels in human FF may lead to downstream events that could potentially determine fertilization and day-3 embryo morphology [
8]. In addition, it was shown that miRNA expression profiling in FF samples could provide biomarkers to identify women with PCOS and to predict blastocyst formation and clinical pregnancy outcome [
17]. However, there have been no studies on the relationships between miRNAs in the FF of endometriosis patients and oocyte quality, fertilization, and early embryo development. In the current study, we hypothesized that miRNAs in the FF of endometriosis patients are associated with oocyte and pre-implantation embryo development.
In this study, we observed significantly lower expression of miR-451 in the FF in women with endometriosis than in healthy women. This finding is consistent with previous reports that miR-451 expression was greatly decreased in eutopic and ectopic endometria in baboons and women with endometriosis [
18]. To assess the role of miR-451 in early embryonic development, miR-451 inhibitor oligonucleotides were used to suppress it in mouse/human oocytes. This study demonstrated that the fertilization, cleavage, and blastocyst rate of mouse/human oocytes were strongly influenced by the knockdown of miR-451, which further suggested that miR-451 has a significant impact on embryo development potential. Our conclusion differs from that of previous research [
19] in which loss-of-function mediated by a lentiviral miR-451 sponge vector (LV-miR-451 sponge) or miR-451 inhibitor reduced the number of embryo implantations but had little effect on fertilization [
19]. However, the previous study differed significantly from ours in the experimental methods used. To efficiently decrease miR-451 expression level, we performed microinjection of the miR-451 inhibitor into the cytoplasm of mouse/human oocytes, whereas Li et al. injected the cornu uteri of an experimental mouse model with LV-miR-451 sponge or the negative control vector.
Although some studies have suggested that miRNA function is repressed in oocytes [
20,
21], others have reported an association between altered miRNA expression levels and both oocyte development and early embryogenesis [
8,
9,
22,
23]. miRNAs play key roles in numerous signalling pathways, and there is evidence for roles of miR-451 in the regulation of multiple signalling pathways, including the Wnt signalling, AMPK signalling, and IL-6R-STAT3 pathways [
24,
25]. Furthermore, several studies have reported that the components of the Wnt signalling pathway are involved in ovarian folliculogenesis [
26] and pre-implantation embryogenesis, including fertilization [
27], embryonic development [
28], trophectoderm specification in human blastocysts [
29], promotion of blastocyst hatching [
30], and embryo implantation [
31]. In addition, it has been suggested that the Wnt signalling pathway is regulated by miR-451 [
32]. These observations indicate that the Wnt signalling pathway plays a role in early pre-implantation embryogenesis, and we postulate that this may be altered upon miR-451 downregulation.
Therefore, we analysed the expression of 12 genes associated with the Wnt signalling pathway in treated mouse oocytes.
Axin1,
Cdx2,
Ctnnb1,
Wnt3, and
Wnt8b were aberrantly expressed in the miR-451 inhibitor-treated group, indicating the suppression of Wnt signalling pathway. Among the five differentially expressed genes,
Axin1,
Cdx2,
Ctnnb1, and
Wnt3 are crucial in oocyte maturation, fertilization, and early embryogenesis [
29,
33‐
35].
Previous evidence suggests that Cdx2 is important for proper pre-implantation embryogenesis, owing to its role in the specification of the trophectoderm lineage from the morula stage [
35]. Knockdown of maternal and zygotic Cdx2 was found to significantly reduce the developmental potential of mouse oocytes and increase cell death from the morula stage onward [
35]. These results suggest that Cdx2 downregulation after the micro-injection of oocytes with miR-451 inhibitor may reduce the developmental potential of mouse oocytes to the blastocyst stage, thus increasing cell death and inhibiting the specification of the trophectoderm lineage. Similar to Cdx2, Ctnnb1 was downregulated in the miR-451 inhibitor-treated group. The inhibition of Ctnnb1 from the zygote stage has been found to reduce blastocyst diameter and number and partially increase embryo fragmentation [
33,
36]. In the present study, the downregulation of Ctnnb1 after oocyte injection with miR-451 inhibitor led to decreased cell number in the embryo and increased embryo fragmentation. Furthermore, this downregulation might have affected the day 3 embryo cell number and fragmentation in patients receiving ICSI, whose FF had relatively low levels of miR-451.
The present investigation has several limitations. First, all the patients included had stage III/IV endometriosis; thus, further validation of our results in patients with early stages of the disease is necessary. Second, the human MII oocytes used in this study had been matured in vitro from the MI stage. The fact that these oocytes were exposed to gonadotrophic stimulation but failed to mature in vivo might reflect their low quality; this should be considered when drawing conclusions from the data.
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